SERPINA1 iRNA COMPOSITIONS AND METHODS OF USE THEREOF

ABSTRACT

The invention relates to RNAi agents, e.g., double-stranded RNAi agents, targeting the Serpina1 gene, and methods of using such RNAi agents to inhibit expression of Serpina1 and methods of treating subjects having a Serpina1 associated disease, such as a liver disorder.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/284,745, filed on May 22, 2014, which claims the benefit of priorityto U.S. Provisional Application No. 61/826,125, filed on May 22, 2013,U.S. Provisional Application No. 61/898,695, filed on Nov. 1, 2013, U.S.Provisional Application No. 61/979,727, filed on Apr. 15, 2014, and U.S.Provisional Application No. 61/989,028, filed on May 6, 2014. Thisapplication is related to U.S. Provisional Application No. 61/561,710,filed on Nov. 18, 2011, and PCT/US2012/065601, filed on Nov. 16, 2012.The entire contents of each of the foregoing applications are herebyincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 3, 2017, isnamed 121301_00606_Seq_Listing.txt and is 199,243 bytes in size.

BACKGROUND OF THE INVENTION

Serpina1 encodes alpha-1-antitrypsin which predominantly complexes withand inhibits the activity of neutrophil elastase produced byhepatocytes, mononuclear monocytes, alveolar macrophages, enterocytes,and myeloid cells. Subjects having variations in one or both copies ofthe Serpina1 gene may suffer from alpha-1-antitrypsin deficiency and areat risk of developing pulmonary emphysema and/or chronic liver diseasedue to greater than normal elastase activity in the lungs and liver.

In affected subjects, the deficiency in alpha-1-antitrypsin is adeficiency of wild-type, functional alpha-1-antitrypsin. In some cases,a subject having a variation in one or both copies of the Serpina1 geneis carrying a null allele. In other cases, a subject having a variationin one or both copies of the Serpina1 gene is carrying a deficientallele.

For example, a subject having a deficient allele of Serpina1, such asthe PIZ allele, may be producing misfolded proteins which cannot beproperly transported from the site of synthesis to the site of actionwithin the body. Such subjects are typically at risk of developing lungand/or liver disease. Subjects having a Serpina1 null allele, such asthe PINULL (Granite Falls), are typically only at risk of developinglung disease.

Liver disease resulting from alpha-1 antitrypsin deficiency is theresult of variant forms of alpha-1-antitypsin produced in liver cellswhich misfold and are, thus, not readily transported out of the cells.This leads to a buildup of misfolded protein in the liver cells and cancause one or more diseases or disorders of the liver including, but notlimited to, chronic liver disease, liver inflammation, cirrhosis, liverfibrosis, and/or hepatocellular carcinoma.

There are currently very limited options for the treatment of patientswith liver disease arising from alpha-1-antitrypsin deficiency,including hepatitis vaccination, supportive care, and avoidance ofinjurious agents (e.g., alcohol and NSAIDs). Although replacementalpha-1-antitrypsin therapy is available, such treatment has no impactliver disease in these subjects and, although liver transplantation maybe effective, it is a difficult, expensive and risky procedure and liverorgans are not readily available.

Accordingly, there is a need in the art for effective treatments forSerpina1-associated diseases, such as a chronic liver disease, liverinflammation, cirrhosis, liver fibrosis, and/or hepatocellularcarcinoma.

SUMMARY OF THE INVENTION

As described in more detail below, disclosed herein are compositionscomprising agents, e.g., single-stranded and double-strandedpolynucleotides, e.g., RNAi agents, e.g., double-stranded iRNA agents,targeting Serpina1. Also disclosed are methods using the compositions ofthe invention for inhibiting Serpina1 expression and for treatingSerpina1 associated diseases, e.g., chronic liver disease, liverinflammation, cirrhosis, liver fibrosis, and/or hepatocellularcarcinoma.

Accordingly, in one aspect, the present invention provides doublestranded RNAi agents for inhibiting expression of Serpina1 in a cell.The double stranded RNAi agents comprise a sense strand and an antisensestrand forming a double-stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from any one of the nucleotide sequences of SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, and theantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from any one of the nucleotide sequencesof SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, or SEQ ID NO:25, wherein substantially all of the nucleotides ofthe sense strand and substantially all of the nucleotides of theantisense strand are modified nucleotides, and wherein the sense strandis conjugated to a ligand attached at the 3′-terminus.

In one embodiment, one of the 3 nucleotide differences in the nucleotidesequence of the antisense strand is a nucleotide mismatch in the seedregion of the antisense strand. In one embodiment, the antisense strandcomprises a universal base at the mismatched nucleotide.

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand are modified nucleotides.

In one embodiment, the sense strand and the antisense strand comprise aregion of complementarity which comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of thesequences listed in any one of Tables 1, 2, 5, 7, 8, and 9.

In one embodiment, at least one of the modified nucleotides is selectedfrom the group consisting of a 3′-terminal deoxy-thymine (dT)nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anabasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modifiednucleotide, a morpholino nucleotide, a phosphoramidate, a non-naturalbase comprising nucleotide, a nucleotide comprising a5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or a dodecanoic acid bisdecylamide group.

In one embodiment, at least one strand comprises a 3′ overhang of atleast 1 nucleotide.

In another embodiment, at least one strand comprises a 3′ overhang of atleast 2 nucleotides.

In another aspect, the present invention provides RNAi agents, e.g.,double-stranded RNAi agents, capable of inhibiting the expression ofSerpina1 in a cell, wherein the double stranded RNAi agent comprises asense strand substantially complementary to an antisense strand, whereinthe antisense strand comprises a region substantially complementary topart of an mRNA encoding Serpina1, wherein each strand is about 14 toabout 30 nucleotides in length, wherein the double stranded RNAi agentis represented by formula (III):

(III) sense: 5′n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not bepresent, independently represents an overhang nucleotide;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In one embodiment, Na′ comprises 1-25 nucleotides, and wherein one ofthe 1-25 nucleotides at one of positions 2-9 from the 5′ end is anucleotide mismatch. In one embodiment, the mismatched base is auniversal base.

In one embodiment, i is 0; j is 0; i is 1; j is 1; both i and j are 0;or both i and j are 1. In another embodiment, k is 0; 1 is 0; k is 1; 1is 1; both k and 1 are 0; or both k and 1 are 1.

In one embodiment, XXX is complementary to X′X′X′, YYY is complementaryto Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′.

In one embodiment, YYY motif occurs at or near the cleavage site of thesense strand.

In one embodiment, Y′Y′Y′ motif occurs at the 11, 12 and 13 positions ofthe antisense strand from the 5′-end.

In one embodiment, Y′ is 2′-O-methyl.

In one embodiment, formula (III) is represented by formula (IIIa):

(IIIa) sense: 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′ antisense: 3′n_(p′)-N_(a′)-Y′Y′Y′-N_(a′)-n_(q′) 5′.

In another embodiment, formula (III) is represented by formula (IIIb):

(IIIb) sense: 5′ n_(p)-N_(a)-Y Y Y-N_(b)-Z Z Z-N_(a)-nq 3′ antisense: 3′n_(p′)-N_(a′)-Y′Y′Y′-N_(b′)-Z′Z′Z′-N_(a′)-n_(q′) 5′

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides.

In yet another embodiment, formula (III) is represented by formula(IIIc):

(IIIc) sense: 5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(a)-n_(q) 3′ antisense:3′ n_(p′)-N_(a′)-X′X′X′-N_(b′)-Y′Y′Y′-N_(a′)-n_(q′) 5′

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides.

In one embodiment, formula (III) is represented by formula (IIId):

(IIId) sense: 5′n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ antisense: 3′n_(p′)-N_(a′)-X′X′X′-N_(b′)-Y′Y′Y′-N_(b′)-Z′Z′Z′-N_(a′)-n_(q′) 5′

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides and eachN_(a) and N_(a)′ independently represents an oligonucleotide sequencecomprising 2-10 modified nucleotides.

In one embodiment, the double-stranded region is 15-30 nucleotide pairsin length. In another embodiment, the double-stranded region is 17-23nucleotide pairs in length. In yet another embodiment, thedouble-stranded region is 17-25 nucleotide pairs in length. In oneembodiment, the double-stranded region is 23-27 nucleotide pairs inlength. In another embodiment, the double-stranded region is 19-21nucleotide pairs in length. In another embodiment, the double-strandedregion is 21-23 nucleotide pairs in length. In one embodiment, eachstrand has 15-30 nucleotides. In another embodiment, each strand has19-30 nucleotides.

In one embodiment, the modifications on the nucleotides are selectedfrom the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl,and combinations thereof. In another embodiment, the modifications onthe nucleotides are 2′-O-methyl or 2′-fluoro modifications.

In one embodiment, the ligand is one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker. In another embodiment,the ligand is

In one embodiment, the ligand is attached to the 3′ end of the sensestrand.

In one embodiment, the RNAi agent is conjugated to the ligand as shownin the following schematic

wherein X is O or S. In a specific embodiment, X is O.

In one embodiment, the agent further comprises at least onephosphorothioate or methylphosphonate internucleotide linkage.

In one embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 3′-terminus of one strand. In oneembodiment, the strand is the antisense strand. In another embodiment,the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 5′-terminus of one strand. In oneembodiment, the strand is the antisense strand. In another embodiment,the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the both the 5′- and 3′-terminus of onestrand. In one embodiment, the strand is the antisense strand.

In one embodiment, the RNAi agent comprises 6-8 phosphorothioateinternucleotide linkages. In one embodiment, the antisense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus and two phosphorothioate internucleotide linkages at the3′-terminus, and the sense strand comprises at least twophosphorothioate internucleotide linkages at either the 5′-terminus orthe 3′-terminus.

In one embodiment, the base pair at the 1 position of the 5′-end of theantisense strand of the duplex is an AU base pair.

In one embodiment, the Y nucleotides contain a 2′-fluoro modification.

In one embodiment, the Y′ nucleotides contain a 2′-O-methylmodification.

In one embodiment, p′>0. In another embodiment, p′=2.

In one embodiment, q′=0, p=0, q=0, and p′ overhang nucleotides arecomplementary to the target mRNA. In another embodiment, q′=0, p=0, q=0,and p′ overhang nucleotides are non-complementary to the target mRNA.

In one embodiment, the sense strand has a total of 21 nucleotides andthe antisense strand has a total of 23 nucleotides.

In one embodiment, at least one n_(p)′ is linked to a neighboringnucleotide via a phosphorothioate linkage.

In one embodiment, all n_(p)′ are linked to neighboring nucleotides viaphosphorothioate linkages.

In one embodiment, the RNAi agent is selected from the group of RNAiagents listed in any one of Tables 1, 2, 5, 7, 8, and 9.

In one embodiment, the RNAi agent is selected from the group consistingof AD-58681, AD-59054, AD-61719, and AD-61444.

In another aspect, the present invention provides double stranded RNAiagent for inhibiting expression of Serpina1 in a cell. The doublestranded RNAi agents comprise a sense strand and an antisense strandforming a double stranded region, wherein the sense strand comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom any one of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, and the antisensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from any one of the nucleotide sequences of SEQ IDNO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ IDNO:25, wherein substantially all of the nucleotides of the sense strandcomprise a modification selected from the group consisting of a2′-O-methyl modification and a 2′-fluoro modification, wherein the sensestrand comprises two phosphorothioate internucleotide linkages at the5′-terminus, wherein substantially all of the nucleotides of theantisense strand comprise a modification selected from the groupconsisting of a 2′-O-methyl modification and a 2′-fluoro modification,wherein the antisense strand comprises two phosphorothioateinternucleotide linkages at the 5′-terminus and two phosphorothioateinternucleotide linkages at the 3′-terminus, and wherein the sensestrand is conjugated to one or more GalNAc derivatives attached througha branched bivalent or trivalent linker at the 3′-terminus.

In one embodiment, one of the 3 nucleotide differences in the nucleotidesequence of the antisense strand is a nucleotide mismatch in the seedregion of the antisense strand. In one embodiment, the antisense strandcomprises a universal base at the mismatched nucleotide.

In one embodiment, all of the nucleotides of said sense strand and allof the nucleotides of said antisense strand comprise a modification.

In another aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agents, capable of inhibiting the expression ofSerpina1 in a cell, wherein the double stranded RNAi agent comprises asense strand substantially complementary to an antisense strand, whereinthe antisense strand comprises a region substantially complementary topart of an mRNA encoding Serpina1, wherein each strand is about 14 toabout 30 nucleotides in length, wherein the double stranded RNAi agentis represented by formula (III):

(III) sense: 5′n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not bepresent independently represents an overhang nucleotide;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In yet another aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agents, capable of inhibiting the expression ofSerpina1 in a cell, wherein the double stranded RNAi agent comprises asense strand substantially complementary to an antisense strand, whereinthe antisense strand comprises a region substantially complementary topart of an mRNA encoding Serpina1, wherein each strand is about 14 toabout 30 nucleotides in length, wherein the double stranded RNAi agentis represented by formula (III):

(III) sense: 5′n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In a further aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agents, capable of inhibiting the expression ofSerpina1 in a cell, wherein the double stranded RNAi agent comprises asense strand substantially complementary to an antisense strand, whereinthe antisense strand comprises a region substantially complementary topart of an mRNA encoding Serpina1, wherein each strand is about 14 toabout 30 nucleotides in length, wherein the double stranded RNAi agentis represented by formula (III):

(III) sense: 5′n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In another aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agents capable of inhibiting the expression ofSerpina1 in a cell, wherein the double stranded RNAi agent comprises asense strand substantially complementary to an antisense strand, whereinthe antisense strand comprises a region substantially complementary topart of an mRNA encoding Serpina1, wherein each strand is about 14 toabout 30 nucleotides in length, wherein the double stranded RNAi agentis represented by formula (III):

(III) sense: 5′n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′;

wherein the sense strand comprises at least one phosphorothioatelinkage; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In yet another aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agents, capable of inhibiting the expression ofSerpina1 in a cell, wherein the double stranded RNAi agent comprises asense strand substantially complementary to an antisense strand, whereinthe antisense strand comprises a region substantially complementary topart of an mRNA encoding Serpina1, wherein each strand is about 14 toabout 30 nucleotides in length, wherein the double stranded RNAi agentis represented by formula (III):

(IIIa) sense: 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′ antisense: 3′n_(p′)-N_(a′)-Y′Y′Y′-N_(a′)-n_(q′) 5′.

wherein:

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

YYY and Y′Y′Y′ each independently represent one motif of three identicalmodifications on three consecutive nucleotides, and wherein themodifications are 2′-O-methyl or 2′-fluoro modifications;

wherein the sense strand comprises at least one phosphorothioatelinkage; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In one embodiment, Na′ comprises 1-25 nucleotides, and wherein one ofthe 1-25 nucleotides at one of positions 2-9 from the 5′ end is anucleotide mismatch. In one embodiment, the mismatched base is auniversal base.

The present invention also provides cells, vectors, host cells, andpharmaceutical compositions comprising the double stranded RNAi agentsof the invention.

In one embodiment, the present invention provides RNAi agent selectedfrom the group of RNAi agents listed in any one of Tables 1, 2, 5, 7, 8,and 9.

The present invention also provides a composition comprising a modifiedantisense polynucleotide agent. The agent is capable of inhibiting theexpression of Serpina1 in a cell, and comprises a sequence complementaryto a sense sequence selected from the group of the sequences listed inany one of Tables 1, 2, 5, 7, 8, and 9, wherein the polynucleotide isabout 14 to about 30 nucleotides in length.

In another aspect, the present invention provides a cell containing thedouble stranded RNAi agent of the invention.

In some embodiments, the RNAi agent is administered using apharmaceutical composition.

In preferred embodiments, the RNAi agent is administered in a solution.In some such embodiments, the siRNA is administered in an unbufferedsolution. In one embodiment, the siRNA is administered in water. Inother embodiments, the siRNA is administered with a buffer solution,such as an acetate buffer, a citrate buffer, a prolamine buffer, acarbonate buffer, or a phosphate buffer or any combination thereof. Insome embodiments, the buffer solution is phosphate buffered saline(PBS).

In one embodiment, the pharmaceutical compositions further comprise alipid formulation. In one aspect, the present invention provides methodsof inhibiting Serpina1 expression in a cell. The methods includecontacting the cell with an RNAi agent, e.g., a double stranded RNAiagent, composition, vector, or a pharmaceutical composition of theinvention; and maintaining the cell produced in step (a) for a timesufficient to obtain degradation of the mRNA transcript of a Serpina1gene, thereby inhibiting expression of the Serpina1 gene in the cell.

In one embodiment, the cell is within a subject.

In one embodiment, the subject is a human.

In one embodiment, the Serpina1 expression is inhibited by at leastabout 30% 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or95%.

In another aspect, the present invention provides methods of treating asubject having a Serpina1 associated disease. The methods includeadministering to the subject a therapeutically effective amount of anRNAi agent, e.g., a double stranded RNAi agent, composition, vector, ora pharmaceutical composition of the invention, thereby treating thesubject.

In another aspect, the present invention provides methods of treating asubject having a Serpina1-associated disorder. The methods includesubcutaneously administering to the subject a therapeutically effectiveamount of a double stranded RNAi agent, wherein the double stranded RNAiagent comprises a sense strand and an antisense strand forming a doublestranded region, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, and the antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from any one of the nucleotide sequences of SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25,wherein substantially all of the nucleotides of the antisense strandcomprise a modification selected from the group consisting of a2′-O-methyl modification and a 2′-fluoro modification, wherein theantisense strand comprises two phosphorothioate internucleotide linkagesat the 5′-terminus and two phosphorothioate internucleotide linkages atthe 3′-terminus, wherein substantially all of the nucleotides of thesense strand comprise a modification selected from the group consistingof a 2′-O-methyl modification and a 2′-fluoro modification, wherein thesense strand comprises two phosphorothioate internucleotide linkages atthe 5′-terminus and, wherein the sense strand is conjugated to one ormore GalNAc derivatives attached through a branched bivalent ortrivalent linker at the 3′-terminus, thereby treating the subject.

In one embodiment, one of the 3 nucleotide differences in the nucleotidesequence of the antisense strand is a nucleotide mismatch in the seedregion of the antisense strand. In one embodiment, the antisense strandcomprises a universal base at the mismatched nucleotide.

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand comprise a modification.

In one embodiment, the Serpina1 associated disease is a liver disorder,e.g., chronic liver disease, liver inflammation, cirrhosis, liverfibrosis, and/or hepatocellular carcinoma

In one embodiment, the administration of the RNAi agent to the subjectresults in a decrease in liver cirrhosis, fibrosis and/or Serpina1protein accumulation in the liver. In another embodiment, theadministration of the RNAi agent to the subject results, e.g., furtherresults, in a decrease in lung inflammation.

In one embodiment, the subject is a human.

In one embodiment, the RNAi agent, e.g., double stranded RNAi agent, isadministered at a dose of about 0.01 mg/kg to about 10 mg/kg, about 0.5mg/kg to about 50 mg/kg, about 10 mg/kg to about 30 mg/kg, about 10mg/kg to about 20 mg/kg, about 15 mg/kg to about 20 mg/kg, about 15mg/kg to about 25 mg/kg, about 15 mg/kg to about 30 mg/kg, or about 20mg/kg to about 30 mg/kg.

In one embodiment, the RNAi agent, e.g., double stranded RNAi agent, isadministered subcutaneously or intravenously.

In yet another aspect, the present invention provides methods ofinhibiting development of hepatocellular carcinoma in a subject having aSerpina1 deficiency variant. The methods include administering to thesubject a therapeutically effective amount of an RNAi agent, e.g., adouble stranded RNAi agent, composition, vector, or a pharmaceuticalcomposition of the invention, thereby inhibiting the development ofhepatocellular carcinoma in the subject.

In another aspect, the present invention provides methods of inhibitingdevelopment of hepatocellular carcinoma in a subject having a Serpina1deficiency variant. The methods include subcutaneously administering tothe subject a therapeutically effective amount of a double stranded RNAiagent, wherein the double stranded RNAi agent comprises a sense strandand an antisense strand forming a double stranded region, wherein thesense strand comprises at least 15 contiguous nucleotides differing byno more than 3 nucleotides from any one of the nucleotide sequences ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ IDNO:11, and the antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of thenucleotide sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, or SEQ ID NO:25, wherein substantially all of thenucleotides of the antisense strand comprise a modification selectedfrom the group consisting of a 2′-O-methyl modification and a 2′-fluoromodification, wherein the antisense strand comprises twophosphorothioate internucleotide linkages at the 5′-terminus and twophosphorothioate internucleotide linkages at the 3′-terminus, whereinsubstantially all of the nucleotides of the sense strand comprise amodification selected from the group consisting of a 2′-O-methylmodification and a 2′-fluoromodification, wherein the sense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus and, wherein the sense strand is conjugated to one or moreGalNAc derivatives attached through a branched bivalent or trivalentlinker at the 3′-terminus, thereby inhibiting development ofhepatocellular carcinoma in the subject having a Serpina1 deficiencyvariant.

In one embodiment, one of the 3 nucleotide differences in the nucleotidesequence of the antisense strand is a nucleotide mismatch in the seedregion of the antisense strand. In one embodiment, the antisense strandcomprises a universal base at the mismatched nucleotide.

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand comprise a modification.

In one embodiment, the subject is a primate or rodent. In anotherembodiment, the subject is a human.

In one embodiment, the RNAi agent, e.g., double stranded RNAi agent, isadministered at a dose of about 0.01 mg/kg to about 10 mg/kg or about0.5 mg/kg to about 50 mg/kg. In another embodiment, the double strandedRNAi agent is administered at a dose of about 10 mg/kg to about 30mg/kg.

In one embodiment, the RNAi agent, e.g., double stranded RNAi agent, isadministered at a dose of about 3 mg/kg. In another embodiment, thedouble stranded RNAi agent is administered at a dose of about 10 mg/kg.In yet another other embodiment, the double stranded RNAi agent isadministered at a dose of about 0.5 mg/kg twice per week. In yet anotherembodiment, the double stranded RNAi agent is administered at a dose ofabout 10 mg/kg every other week. In yet another embodiment, the doublestranded RNAi agent is administered at a dose of about 0.5 to about 1mg/kg once per week.

In one embodiment, the RNAi agent, e.g., double stranded RNAi agent, isadministered twice per week. In another embodiment, the RNAi agent isadministered every other week.

In one embodiment, the RNAi agent, e.g., double stranded RNAi agent, isadministered subcutaneously or intravenously.

In another aspect, the present invention provides methods for reducingthe accumulation of misfolded Serpina1 in the liver of a subject havinga Serpina1 deficiency variant. The methods include administering to thesubject a therapeutically effective amount of an RNAi agent, e.g., adouble stranded RNAi agent, composition, vector, or a pharmaceuticalcomposition of the invention, thereby reducing the accumulation ofmisfolded Serpina1 in the liver of the subject.

In another aspect, the present invention provides methods of reducingthe accumulation of misfolded Serpina1 in the liver of a subject havinga Serpina1 deficiency variant. The methods include subcutaneouslyadministering to the subject a therapeutically effective amount of adouble stranded RNAi agent, wherein the double stranded RNAi agentcomprises a sense strand and an antisense strand forming a doublestranded region, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, and the antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from any one of the nucleotide sequences of SEQ ID NO: 15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25,wherein substantially all of the nucleotides of the antisense strandcomprise a modification selected from the group consisting of a2′-O-methyl modification and a 2′-fluoromodification, wherein theantisense strand comprises two phosphorothioate internucleotide linkagesat the 5′-terminus and two phosphorothioate internucleotide linkages atthe 3′-terminus, wherein substantially all of the nucleotides of thesense strand comprise a modification selected from the group consistingof a 2′-O-methyl modification and a 2′-fluoro modification, wherein thesense strand comprises two phosphorothioate internucleotide linkages atthe 5′-terminus and, wherein the sense strand is conjugated to one ormore GalNAc derivatives attached through a branched bivalent ortrivalent linker at the 3′-terminus, thereby reducing the accumulationof misfolded Serpina1 in the liver of the subject having a Serpina1deficiency variant.

In one embodiment, one of the 3 nucleotide differences in the nucleotidesequence of the antisense strand is a nucleotide mismatch in the seedregion of the antisense strand. In one embodiment, the antisense strandcomprises a universal base at the mismatched nucleotide.

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand comprise a modification.

In one embodiment, the subject is a primate or rodent. In anotherembodiment, the subject is a human.

In one embodiment, the RNAi agent, e.g., double stranded RNAi agent, isadministered at a dose of about 0.01 mg/kg to about 10 mg/kg or about0.5 mg/kg to about 50 mg/kg. In another embodiment, the double strandedRNAi agent is administered at a dose of about 10 mg/kg to about 30mg/kg.

In one embodiment, the RNAi agent, e.g., double stranded RNAi agent, isadministered subcutaneously or intravenously.

The present invention is further illustrated by the following detaileddescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the in vivo efficacy and duration ofresponse for the indicated siRNAs in transgenic mice expressing theZ-AAT form of human AAT.

FIGS. 2A-2B depict in vivo efficacy of five siRNAs with low IC50 values.Transgenic mice expressing the human Z-AAT allele were injected with 10mg/kg siRNA duplex on day 0 and serum human AAT was followed for 21 dayspost dose (FIG. 2A). Each point represents an average of three mice andthe error bars reflect the standard deviation. FIG. 2B depicts hAAT mRNAlevels in liver normalized to GAPDH for each group. The bars reflect theaverage and the error bars reflect the standard deviation.

FIGS. 3A-3C depict durable AAT suppression in a dose responsive manner.FIG. 3A specifically depicts the efficacy curve showing maximumknock-down of serum hAAT protein levels achieved at different doses ofAD-59054 subcutaneously administered to transgenic mice. Each point isan average of three animals and the error bars represent the standarddeviation. The duration of knock-down after a single dose of AAT siRNAat 0.3, 1, 3 or 10 mg/kg is shown in FIG. 3B. The hAAT levels werenormalized to the average of three prebleeds for each animal. The PBSgroup serves as the control to reflect the variability in the serum hAATlevels. Each data point is an average of three animals and the errorbars reflect the standard deviation. In FIG. 3C, animals wereadministered AD-59054 at a dose of 0.5 mg/kg twice a week. Each datapoint is an average relative serum hAAT from four animals and the errorbars reflect the standard deviation.

FIGS. 4A-4D depict decreased tumor incidence with reduction in Z-AAT.FIG. 4A depicts the study design whereby aged mice with fibrotic liverswere dosed subcutaneously once every other week (Q2W) with PBS or 10mg/kg siRNA duplex AD58681 for 11 doses and sacrificed 7 days after thelast dose. FIG. 4B shows liver levels of hAAT mRNA in control andtreated groups. FIG. 4C shows liver levels of Col1a2 mRNA in control andtreated groups. FIG. 4D depicts liver levels of PtPrc mRNA in controland treated groups.

FIGS. 5A-5C depict decreased tumor incidence with reduction in Z-AAT.Serum samples were collected from mice treated according to the studydesign of FIG. 4A to monitor the extent of hAAT suppression. FIG. 5Adepicts serum hAAT protein levels after the first dose. FIG. 5B and FIG.5C depict PAS staining of liver sections from two littermates treatedwith either PBS or AAT siRNA. The darker colored dots represent theglobules or Z-AAT aggregates.

FIG. 6 depicts the in vivo efficacy of the indicated compounds.

FIGS. 7A and 7B are graphs depicting the duration of knock-down of AATin non-human primates after a single dose of AD-59054, AD-61719, orAD-61444 at a dose of 1 mg/kg (7A) or 3 mg/kg (7B). Each data point isan average of three animals and the error bars reflect the standarddeviation.

FIG. 8A shows the nucleotide sequence of Homo sapiens Serpina1,transcript variant 1 (SEQ ID NO:1); FIG. 8B shows the nucleotidesequence of Homo sapiens Serpina1, transcript variant 3 (SEQ ID NO:2);FIG. 8C shows the nucleotide sequence of Homo sapiens Serpina1,transcript variant 2 (SEQ ID NO:3); FIG. 8D shows the nucleotidesequence of Homo sapiens Serpina1, transcript variant 4 (SEQ ID NO:4);FIG. 8E shows the nucleotide sequence of Homo sapiens Serpina1,transcript variant 5 (SEQ ID NO:5); FIG. 8F shows the nucleotidesequence of Homo sapiens Serpina1, transcript variant 6 (SEQ ID NO:6);FIG. 8G shows the nucleotide sequence of Homo sapiens Serpina1,transcript variant 7 (SEQ ID NO:7); FIG. 8H shows the nucleotidesequence of Homo sapiens Serpina1, transcript variant 8 (SEQ ID NO:8);FIG. 8I shows the nucleotide sequence of Homo sapiens Serpina1,transcript variant 9 (SEQ ID NO:9); FIG. 8J shows the nucleotidesequence of Homo sapiens Serpina1, transcript variant 10 (SEQ ID NO:10);FIG. 8K shows the nucleotide sequence of Homo sapiens Serpina1,transcript variant 11 (SEQ ID NO:11); FIG. 8L shows the nucleotidesequence of Macaca mulatta Serpina1 (SEQ ID NO:12); FIG. 8M shows thenucleotide sequence of Macaca mulatta Serpina1, transcript variant 6(SEQ ID NO:13); FIG. 8N shows the nucleotide sequence of Macaca mulattaSerpina1, transcript variant 4 (SEQ ID NO: 14); FIG. 8O shows thereverse complement of SEQ ID NO:1 (SEQ ID NO:15); FIG. 8P shows thereverse complement of SEQ ID NO:2 (SEQ ID NO:16); FIG. 8Q shows thereverse complement of SEQ ID NO:3 (SEQ ID NO:17); FIG. 8R shows thereverse complement of SEQ ID NO:4 (SEQ ID NO:18); FIG. 8S shows thereverse complement of SEQ ID NO:5 (SEQ ID NO:19); FIG. 8T shows thereverse complement of SEQ ID NO:6 (SEQ ID NO:20); FIG. 8U shows thereverse complement of SEQ ID NO:7 (SEQ ID NO:21); FIG. 8V shows thereverse complement of SEQ ID NO:8 (SEQ ID NO:22); FIG. 8W shows thereverse complement of SEQ ID NO:9 (SEQ ID NO:23); FIG. 8X shows thereverse complement of SEQ ID NO:10 (SEQ ID NO:24); FIG. 8Y shows thereverse complement of SEQ ID NO:11 (SEQ ID NO:25); FIG. 8Z shows thereverse complement of SEQ ID NO:12 (SEQ ID NO:26); FIG. 8AA shows thereverse complement of SEQ ID NO: 13 (SEQ ID NO:27); and FIG. 8AB showsthe reverse complement of SEQ ID NO: 14 (SEQ ID NO:28).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions comprising agents, e.g.,single-stranded and double-stranded oligonucleotides, e.g., RNAi agents,e.g., double-stranded iRNA agents, targeting Serpina1. Also disclosedare methods using the compositions of the invention for inhibitingSerpina1 expression and for treating Serpina1 associated diseases, suchas liver disorders, e.g., chronic liver disease, liver inflammation,cirrhosis, liver fibrosis, and/or hepatocellular carcinoma.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, “Serpina1” refers to the serpin peptidase inhibitor,clade A, member 1 gene or protein. Serpina1 is also known asalpha-1-antitrypsin, α-1-antitrypsin, AAT, protease inhibitor 1, PI,PI1, anti-elastase, and antitrypsin.

The term Serpina1 includes human Serpina1, the amino acid and nucleotidesequence of which may be found in, for example, GenBank Accession Nos.GI:189163524 (SEQ ID NO:1), GI:189163525 (SEQ ID NO:2), GI:189163526(SEQ ID NO:3), GI:189163527 (SEQ ID NO:4), GI:189163529 (SEQ ID NO:5),GI:189163531 (SEQ ID NO:6), GI:189163533 (SEQ ID NO:7), GI:189163535(SEQ ID NO:8), GI:189163537 (SEQ ID NO:9), GI:189163539 (SEQ ID NO:10),and/or GI:189163541 (SEQ ID NO:11); rhesus Serpina1, the amino acid andnucleotide sequence of which may be found in, for example, GenBankAccession Nos. GI:402766667 (SEQ ID NO:12), GI:297298519 (SEQ ID NO:13),and/or GI: 297298520 (SEQ ID NO:14); mouse Serpina1, the amino acid andnucleotide sequence of which may be found in, for example, GenBankAccession No. GI:357588423 and/or GI:357588426; and rat, the amino acidand nucleotide sequence of which may be found in, for example, GenBankAccession No. GI:77020249. Additional examples of Serpina1 mRNAsequences are readily available using, e.g., GenBank and OMIM.

Over 120 alleles of Serpina1 have been identified and the “M” allelesare considered the wild-type or “normal” allele (e.g., “PIM1-ALA213”(also known as PI, M1A), “PIM1-VAL213” (also known as PI, MIV), “PIM2”,“PIM3”, and PIM4”). Additional variants may be found in, for example,the A(1)ATVar database (see, e.g., Zaimidou, S., et al. (2009) HumMutat. 230(3):308-13 and www.goldenhelix.org/A1ATVar).

As used herein, the term “Serpina1 deficiency allele” refers to avariant allele that produces proteins which do not fold properly and mayaggregate intracellularly and are, thus, not properly transported fromthe site of synthesis in the liver to the site of action within thebody.

Exemplary Serpina1 deficiency alleles include, the “Z allele”, the “Sallele”, the “PIM(Malton) allele”, and the “PIM(Procida) allele”.

As used herein, the terms “Z allele”, “PIZ” and “Z-AAT” refer to avariant allele of Serpina1 in which the amino acid at position 342 ofthe protein is changed from a glutamine to a lysine as a result of therelevant codon being changed from GAG to AAG. A subject homozygous for aZ allele can be referred to as “PIZZ.” Z-AAT mutations account for 95%of Serpina1 deficiency patients and are estimated to be present in100,000 Americans and about 3 million individuals worldwide. The Zallele reaches polymorphic frequencies in Caucasians and is rare orabsent in Asians and blacks. The homozygous ZZ phenotype is associatedwith a high risk of both emphysema and liver disease. Z-AAT protein doesnot fold correctly in the endoplasmic reticulum, leading to loop-sheetpolymers which aggregate and reduce secretion, elicitation of theunfolded protein response, apoptosis, endoplasmic reticulum overloadresponse, autophagy, mitochondrial stress, and altered hepatocytefunction.

As used herein, the terms “PIM(Malton)” and “M(Malton)-AAT” refer to avariant allele of Serpina1 in which one of the adjacent phenylalanineresidues at position 51 or 52 of the mature protein is deleted. Deletionof this one amino acid shortens one strand of the beta-sheet, B6,preventing normal processing and secretion in the liver which isassociated with hepatocyte inclusions and impaired secretion of theprotein from the liver.

As used herein, the term “PIS” refers to a variant allele of Serpina1 inwhich a glutamic acid at position 264 is substituted with valine.Although the majority of this variant protein is degradedintracellularly, there is a high frequency of the PIS allele in theCaucasian population and, thus, compound heterozygotes with a Z or nullallele are frequent.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a Serpina1 gene, including mRNA that is a product of RNA processingof a primary transcription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.“T” and “dT” are used interchangeably herein and refer to adeoxyribonucleotide wherein the nucleobase is thymine, e.g.,deoxyribothymine, 2′-deoxythymidine or thymidine. However, it will beunderstood that the term “ribonucleotide” or “nucleotide” or“deoxyribonucleotide” can also refer to a modified nucleotide, asfurther detailed below, or a surrogate replacement moiety. The skilledperson is well aware that guanine, cytosine, adenine, and uracil may bereplaced by other moieties without substantially altering the basepairing properties of an oligonucleotide comprising a nucleotide bearingsuch replacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base may base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine may be replaced in the nucleotide sequences of theinvention by a nucleotide containing, for example, inosine. Sequencescomprising such replacement moieties are embodiments of the invention.The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent”as used interchangeably herein, refer to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.iRNA directs the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The iRNA modulates, e.g., inhibits,the expression of Serpina1 in a cell, e.g., a cell within a subject,such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a singlestranded RNA that interacts with a target RNA sequence, e.g., a Serpina1target mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory, it is believed that long double strandedRNA introduced into cells is broken down into siRNA by a Type IIIendonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485).Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect theinvention relates to a single stranded RNA (siRNA) generated within acell and which promotes the formation of a RISC complex to effectsilencing of the target gene, i.e., a Serpina1 gene. Accordingly, theterm “siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded siRNAthat is introduced into a cell or organism to inhibit a target mRNA.Single-stranded RNAi agents bind to the RISC endonuclease Argonaute 2,which then cleaves the target mRNA. The single-stranded siRNAs aregenerally 15-30 nucleotides and are chemically modified. The design andtesting of single-stranded siRNAs are described in U.S. Pat. No.8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entirecontents of each of which are hereby incorporated herein by reference.Any of the antisense nucleotide sequences described herein may be usedas a single-stranded siRNA as described herein or as chemically modifiedby the methods described in Lima et al., (2012) Cell 150:883-894.

In yet another embodiment, the present invention providessingle-stranded antisense oligonucleotide molecules targeting Serpina1.A “single-stranded antisense oligonucleotide molecule” is complementaryto a sequence within the target mRNA (i.e., Serpina1). Single-strandedantisense oligonucleotide molecules can inhibit translation in astoichiometric manner by base pairing to the mRNA and physicallyobstructing the translation machinery, see Dias, N. et al., (2002) MolCancer Ther 1:347-355. Alternatively, the single-stranded antisenseoligonucleotide molecules inhibit a target mRNA by hydridizing to thetarget and cleaving the target through an RNaseH cleavage event. Thesingle-stranded antisense oligonucleotide molecule may be about 10 toabout 30 nucleotides in length and have a sequence that is complementaryto a target sequence. For example, the single-stranded antisenseoligonucleotide molecule may comprise a sequence that is at least about10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguousnucleotides from any one of the antisense nucleotide sequences describedherein, e.g., the sequences provided in any one of Tables, 1, 2, 5, 7,8, or 9 or bind any of the target sites described herein. Thesingle-stranded antisense oligonucleotide molecules may comprisemodified RNA, DNA, or a combination thereof.

In another embodiment, an “iRNA” for use in the compositions, uses, andmethods of the invention is a double-stranded RNA and is referred toherein as a “double stranded RNAi agent,” “double-stranded RNA (dsRNA)molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary nucleicacid strands, referred to as having “sense” and “antisense” orientationswith respect to a target RNA, i.e., a Serpina1 gene. In some embodimentsof the invention, a double-stranded RNA (dsRNA) triggers the degradationof a target RNA, e.g., an mRNA, through a post-transcriptionalgene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNAmolecule are ribonucleotides, but as described in detail herein, each orboth strands can also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, an “RNAi agent” may include ribonucleotides withchemical modifications; an RNAi agent may include substantialmodifications at multiple nucleotides. Such modifications may includeall types of modifications disclosed herein or known in the art. Anysuch modifications, as used in a siRNA type molecule, are encompassed by“RNAi agent” for the purposes of this specification and claims.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” Where the two strands are connected covalently by means otherthan an uninterrupted chain of nucleotides between the 3′-end of onestrand and the 5′-end of the respective other strand forming the duplexstructure, the connecting structure is referred to as a “linker.” TheRNA strands may have the same or a different number of nucleotides. Themaximum number of base pairs is the number of nucleotides in theshortest strand of the dsRNA minus any overhangs that are present in theduplex. In addition to the duplex structure, an RNAi agent may compriseone or more nucleotide overhangs.

In one embodiment, an RNAi agent of the invention is a dsRNA of 24-30nucleotides that interacts with a target RNA sequence, e.g., a Serpina1target mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory, long double stranded RNA introduced intocells is broken down into siRNA by a Type III endonuclease known asDicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, aribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pairshort interfering RNAs with characteristic two base 3′ overhangs(Bernstein, et al., (2001) Nature 409:363). The siRNAs are thenincorporated into an RNA-induced silencing complex (RISC) where one ormore helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of anRNAi agent when a 3′-end of one strand of the RNAi agent extends beyondthe 5′-end of the other strand, or vice versa. “Blunt” or “blunt end”means that there are no unpaired nucleotides at that end of the doublestranded RNAi agent, i.e., no nucleotide overhang. A “blunt ended” RNAiagent is a dsRNA that is double-stranded over its entire length, i.e.,no nucleotide overhang at either end of the molecule. The RNAi agents ofthe invention include RNAi agents with nucleotide overhangs at one end(i.e., agents with one overhang and one blunt end) or with nucleotideoverhangs at both ends.

The term “antisense strand” refers to the strand of a double strandedRNAi agent which includes a region that is substantially complementaryto a target sequence (e.g., a human Serpina1 mRNA). As used herein, theterm “region complementary to part of an mRNA encoding Serpina1” refersto a region on the antisense strand that is substantially complementaryto part of a Serpina1 mRNA sequence. Where the region of complementarityis not fully complementary to the target sequence, the mismatches aremost tolerated in the terminal regions and, if present, are generally ina terminal region or regions, e.g., within 8, 7, 6, 5, 4, 3, or 2nucleotides of the 5′ and/or 3′ terminus.

As demonstrated in the working examples below, it has been surprisinglydiscovered that a single nucleotide mismatch in the seed region of theantisense strand of the RNAi agents disclosed herein was tolerated forall bases except C. The “seed region” is the region in the antisensestrand of an RNAi agent responsible for recognition of the target mRNAand corresponds to, for example, nucleotides 2-8 from the 5′ end of theantisense strand. After the seed region anneals, Argonaute then subjectscomplementary mRNA sequences 10 nucleotides from the 5′ end of theincorporated antisense strand to nucleolytic degradation, resulting inthe cleavage of the target mRNA. Accordingly, in one embodiment, theantisense strand of an RNAi agent of the invention comprises a onenucleotide mismatch in the seed region of the antisense strand, e.g., amismatch at any one of positions 2-8 from the 5′-end of the antisensestrand.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. For example, a complementarysequence is sufficient to allow the relevant function of the nucleicacid to proceed, e.g., RNAi. The skilled person will be able todetermine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

Sequences can be “fully complementary” with respect to each when thereis base-pairing of the nucleotides of the first nucleotide sequence withthe nucleotides of the second nucleotide sequence over the entire lengthof the first and second nucleotide sequences. However, where a firstsequence is referred to as “substantially complementary” with respect toa second sequence herein, the two sequences can be fully complementary,or they may form one or more, but generally not more than 4, 3 or 2mismatched base pairs upon hybridization, while retaining the ability tohybridize under the conditions most relevant to their ultimateapplication. However, where two oligonucleotides are designed to form,upon hybridization, one or more single stranded overhangs, suchoverhangs shall not be regarded as mismatches with regard to thedetermination of complementarity. For example, a dsRNA comprising oneoligonucleotide 21 nucleotides in length and another oligonucleotide 23nucleotides in length, wherein the longer oligonucleotide comprises asequence of 21 nucleotides that is fully complementary to the shorteroligonucleotide, may yet be referred to as “fully complementary” for thepurposes described herein.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding Serpina1) including a 5′ UTR, an openreading frame (ORF), or a 3′ UTR. For example, a polynucleotide iscomplementary to at least a part of a Serpina1 mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding Serpina1.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating,” “suppressing” and othersimilar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a Serpina1,” as used herein,includes inhibition of expression of any Serpina1 gene (such as, e.g., amouse Serpina1 gene, a rat Serpina1 gene, a monkey Serpina1 gene, or ahuman Serpina1 gene) as well as variants, (e.g., naturally occurringvariants), or mutants of a Serpina1 gene. Thus, the Serpina1 gene may bea wild-type Serpina1 gene, a variant Serpina1 gene, a mutant Serpina1gene, or a transgenic Serpina1 gene in the context of a geneticallymanipulated cell, group of cells, or organism.

“Inhibiting expression of a Serpina1 gene” includes any level ofinhibition of a Serpina1 gene, e.g., at least partial suppression of theexpression of a Serpina1 gene, such as an inhibition of at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%.at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least about 99%.

The expression of a Serpina1 gene may be assessed based on the level ofany variable associated with Serpina1 gene expression, e.g., Serpina1mRNA level, Serpina1 protein level, or serum AAT levels. Inhibition maybe assessed by a decrease in an absolute or relative level of one ormore of these variables compared with a control level. The control levelmay be any type of control level that is utilized in the art, e.g., apre-dose baseline level, or a level determined from a similar subject,cell, or sample that is untreated or treated with a control (such as,e.g., buffer only control or inactive agent control).

The phrase “contacting a cell with a double stranded RNAi agent,” asused herein, includes contacting a cell by any possible means.Contacting a cell with a double stranded RNAi agent includes contactinga cell in vitro with the RNAi agent or contacting a cell in vivo withthe RNAi agent. The contacting may be done directly or indirectly. Thus,for example, the RNAi agent may be put into physical contact with thecell by the individual performing the method, or alternatively, the RNAiagent may be put into a situation that will permit or cause it tosubsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the RNAi agent. Contacting a cell in vivo may be done, forexample, by injecting the RNAi agent into or near the tissue where thecell is located, or by injecting the RNAi agent into another area, thebloodstream or the subcutaneous space, such that the agent willsubsequently reach the tissue where the cell to be contacted is located.For example, the RNAi agent may contain and/or be coupled to a ligand,e.g., a GalNAc3 ligand, that directs the RNAi agent to a site ofinterest, e.g., the liver. Combinations of in vitro and in vivo methodsof contacting are also possible. In connection with the methods of theinvention, a cell might also be contacted in vitro with an RNAi agentand subsequently transplanted into a subject.

A “patient” or “subject,” as used herein, is intended to include eithera human or non-human animal, preferably a mammal, e.g., a monkey. Mostpreferably, the subject or patient is a human.

A “Serpina1 associated disease,” as used herein, is intended to includeany disease, disorder, or condition associated with the Serpina1 gene orprotein. Such a disease may be caused, for example, by misfolding of aSerpina1 protein, intracellular accumulation of Serpina1 protein (e.g.,misfolded Serpina1 protein), excess production of the Serpina1 protein,by Serpina1 gene variants, Serpina1 gene mutations, by abnormal cleavageof the Serpina1 protein, by abnormal interactions between Serpina1 andother proteins or other endogenous or exogenous substances. A Serpina1associated disease may be a liver disease and/or a lung disease.

A “liver disease”, as used herein, includes a disease, disorder, orcondition affecting the liver and/or its function. A liver disorder canbe the result of accumulation of Serpina1 protein in the liver and/orliver cells. Examples of liver disorders include liver disordersresulting from, viral infections, parasitic infections, geneticpredisposition, autoimmune diseases, exposure to radiation, exposure tohepatotoxic compounds, mechanical injuries, various environmentaltoxins, alcohol, acetaminophen, a combination of alcohol andacetaminophen, inhalation anesthetics, niacin, chemotherapeutics,antibiotics, analgesics, antiemetics and the herbal supplement kava, andcombinations thereof.

For example, a liver disorder associated with Serpina1 deficiency mayoccur more often in subjects with one or more copies of certain alleles(e.g., the PIZ, PiM(Malton), and/or PIS alleles). Without wishing to bebound by theory, it is thought that alleles associated with a greaterrisk of developing an alpha-1 anti-trypsin liver disease encode forms ofSerpina1 which are subject to misfolding and are not properly secretedfrom the hepatocytes. The cellular responses to these misfolded proteinscan include the unfolded protein response (UPR), endoplasmicreticulum-associated degradation (ERAD), apoptosis, ER overloadresponse, autophagy, mitochondrial stress and altered hepatocytefunction. The injuries to the hepatocytes can lead to symptoms such as,but not limited to, inflammation, cholestasis, fibrosis, cirrhosis,prolonged obstructive jaundice, increased transaminases, portalhypertension and/or hepatocellular carcinoma. Without wishing to bebound by theory, the highly variable clinical course of this disease issuggestive of modifiers or “second hits” as contributors to developingsymptoms or progressing in severity.

For example, subjects with a PIZ allele can be more sensitive toHepatitis C infections or alcohol abuse and more likely to develop aliver disorder if exposed to such factors. Additionally cystic fibrosis(CF) subjects carrying the PIZ allele are at greater risk of developingsevere liver disease with portal hypertension. A deficiency of Serpina1can also cause or contribute to the development of early onsetemphysema, necrotizing panniculitis, bronchiectasis, and/or prolongedneonatal jaundice. Some patients having or at risk of having adeficiency of alpha-1-antitrypsin are identified by screening when theyhave family members affected by an alpha-1-antitrypsin deficiency.

Exemplary liver disorders include, but are not limited to, liverinflammation, chronic liver disease, cirrhosis, liver fibrosis,hepatocellular carcinoma, liver necrosis, steatosis, cholestatis and/orreduction and/or loss of hepatocyte function.

“Cirrhosis” is a pathological condition associated with chronic liverdamage that includes extensive fibrosis and regenerative nodules in theliver.

“Fibrosis” is the proliferation of fibroblasts and the formation of scartissue in the liver.

The phrase “liver function” refers to one or more of the manyphysiological functions performed by the liver. Such functions include,but are not limited to, regulating blood sugar levels, endocrineregulation, enzyme systems, interconversion of metabolites (e.g., ketonebodies, sterols and steroids and amino acids); manufacturing bloodproteins such as fibrinogen, serum albumin, and cholinesterase,erythropoietic function, detoxification, bile formation, and vitaminstorage. Several tests to examine liver function are known in the art,including, for example, measuring alanine amino transferase (ALT),alkaline phosphatase, bilirubin, prothrombin, and albumin.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a patientfor treating a Serpina1-associated disease, is sufficient to effecttreatment of the disease (e.g., by diminishing, ameliorating ormaintaining the existing disease or one or more symptoms of disease).The “therapeutically effective amount” may vary depending on the RNAiagent, how the agent is administered, the disease and its severity andthe history, age, weight, family history, genetic makeup, stage ofpathological processes mediated by Serpina1 expression, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the patient to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjectwho does not yet experience or display symptoms of anSerpina1-associated disease, but who may be predisposed to the disease,is sufficient to prevent or ameliorate the disease or one or moresymptoms of the disease. Ameliorating the disease includes slowing thecourse of the disease or reducing the severity of later-developingdisease. The “prophylactically effective amount” may vary depending onthe RNAi agent, how the agent is administered, the degree of risk ofdisease, and the history, age, weight, family history, genetic makeup,the types of preceding or concomitant treatments, if any, and otherindividual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effectiveamount” also includes an amount of an RNAi agent that produces somedesired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment. RNAi gents employed in the methods of thepresent invention may be administered in a sufficient amount to producea reasonable benefit/risk ratio applicable to such treatment.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, urine, lymph,cerebrospinal fluid, ocular fluids, saliva, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In preferred embodiments, a “sample derived from asubject” refers to blood or plasma drawn from the subject. In furtherembodiments, a “sample derived from a subject” refers to liver tissue(or subcomponents thereof) derived from the subject.

II. iRNAs of the Invention

Described herein are improved double-stranded RNAi agents which inhibitthe expression of a Serpina1 gene in a cell, such as a cell within asubject, e.g., a mammal, such as a human having a Serpina1 associateddisease, e.g., a liver disease, e.g., chronic liver disease, liverinflammation, cirrhosis, liver fibrosis, and/or hepatocellularcarcinoma.

Accordingly, the invention provides double-stranded RNAi agents withchemical modifications capable of inhibiting the expression of a targetgene (i.e., a Serpina1 gene) in vivo.

In certain aspects of the invention, substantially all of thenucleotides of an iRNA of the invention are modified. In otherembodiments of the invention, all of the nucleotides of an iRNA of theinvention are modified. iRNAs of the invention in which “substantiallyall of the nucleotides are modified” are largely but not wholly modifiedand can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.

The RNAi agent comprises a sense strand and an antisense strand. Eachstrand of the RNAi agent may range from 12-30 nucleotides in length. Forexample, each strand may be between 14-30 nucleotides in length, 17-30nucleotides in length, 19-30 nucleotides in length, 25-30 nucleotides inlength, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides inlength, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex doublestranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” Theduplex region of an RNAi agent may be 12-30 nucleotide pairs in length.For example, the duplex region can be between 14-30 nucleotide pairs inlength, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs inlength, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs inlength, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs inlength, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs inlength, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs inlength. In another example, the duplex region is selected from 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In one embodiment, the RNAi agent may contain one or more overhangregions and/or capping groups at the 3′-end, 5′-end, or both ends of oneor both strands. The overhang can be 1-6 nucleotides in length, forinstance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides inlength, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2nucleotides in length. The overhangs can be the result of one strandbeing longer than the other, or the result of two strands of the samelength being staggered. The overhang can form a mismatch with the targetmRNA or it can be complementary to the gene sequences being targeted orcan be another sequence. The first and second strands can also bejoined, e.g., by additional bases to form a hairpin, or by othernon-base linkers.

In one embodiment, the nucleotides in the overhang region of the RNAiagent can each independently be a modified or unmodified nucleotideincluding, but no limited to 2′-sugar modified, such as, 2-F,2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo),2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine(m5Ceo), and any combinations thereof. For example, TT can be anoverhang sequence for either end on either strand. The overhang can forma mismatch with the target mRNA or it can be complementary to the genesequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or bothstrands of the RNAi agent may be phosphorylated. In some embodiments,the overhang region(s) contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In one embodiment, the overhang is presentat the 3′-end of the sense strand, antisense strand, or both strands. Inone embodiment, this 3′-overhang is present in the antisense strand. Inone embodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthenthe interference activity of the RNAi, without affecting its overallstability. For example, the single-stranded overhang may be located atthe 3′-terminal end of the sense strand or, alternatively, at the3′-terminal end of the antisense strand. The RNAi may also have a bluntend, located at the 5′-end of the antisense strand (or the 3′-end of thesense strand) or vice versa. Generally, the antisense strand of the RNAihas a nucleotide overhang at the 3′-end, and the 5′-end is blunt. Whilenot wishing to be bound by theory, the asymmetric blunt end at the5′-end of the antisense strand and 3′-end overhang of the antisensestrand favor the guide strand loading into RISC process.

Any of the nucleic acids featured in the invention can be synthesizedand/or modified by methods well established in the art, such as thosedescribed in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, whichis hereby incorporated herein by reference. Modifications include, forexample, end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; and/orbackbone modifications, including modification or replacement of thephosphodiester linkages. Specific examples of iRNA compounds useful inthe embodiments described herein include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In some embodiments, amodified iRNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use iniRNAs, in which both the sugar and the internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, an RNAmimetic that has been shown to have excellent hybridization properties,is referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Representative U.S. patents thatteach the preparation of PNA compounds include, but are not limited to,U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contentsof each of which are hereby incorporated herein by reference. AdditionalPNA compounds suitable for use in the iRNAs of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂-[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂-[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O] _(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as deoxy-thymine (dT), 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

Representative U.S. patents that teach the preparation of locked nucleicacid nucleotides include, but are not limited to, the following: U.S.Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207;7,084,125; and 7,399,845, the entire contents of each of which arehereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double-stranded RNAi agents ofthe invention include agents with chemical modifications as disclosed,for example, in U.S. Provisional Application No. 61/561,710, filed onNov. 18, 2011, or in PCT/US2012/065691, filed on Nov. 16, 2012, theentire contents of each of which are incorporated herein by reference.

As shown herein and in Provisional Application No. 61/561,710, asuperior result may be obtained by introducing one or more motifs ofthree identical modifications on three consecutive nucleotides into asense strand and/or antisense strand of a RNAi agent, particularly at ornear the cleavage site. In some embodiments, the sense strand andantisense strand of the RNAi agent may otherwise be completely modified.The introduction of these motifs interrupts the modification pattern, ifpresent, of the sense and/or antisense strand. The RNAi agent may beoptionally conjugated with a GalNAc derivative ligand, for instance onthe sense strand. The resulting RNAi agents present superior genesilencing activity.

More specifically, it has been surprisingly discovered that when thesense strand and antisense strand of the double-stranded RNAi agent aremodified to have one or more motifs of three identical modifications onthree consecutive nucleotides at or near the cleavage site of at leastone strand of an RNAi agent, the gene silencing activity of the RNAiagent was superiorly enhanced.

In one embodiment, the RNAi agent is a double ended bluntmer of 19nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 7, 8, 9 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In another embodiment, the RNAi agent is a double ended bluntmer of 20nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 8, 9, 10 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In yet another embodiment, the RNAi agent is a double ended bluntmer of21 nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 9, 10, 11 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strandand a 23 nucleotide antisense strand, wherein the sense strand containsat least one motif of three 2′-F modifications on three consecutivenucleotides at positions 9, 10, 11 from the 5′ end; the antisense strandcontains at least one motif of three 2′-O-methyl modifications on threeconsecutive nucleotides at positions 11, 12, 13 from the 5′ end, whereinone end of the RNAi agent is blunt, while the other end comprises a 2nucleotide overhang. Preferably, the 2 nucleotide overhang is at the3′-end of the antisense strand. When the 2 nucleotide overhang is at the3′-end of the antisense strand, there may be two phosphorothioateinternucleotide linkages between the terminal three nucleotides, whereintwo of the three nucleotides are the overhang nucleotides, and the thirdnucleotide is a paired nucleotide next to the overhang nucleotide. Inone embodiment, the RNAi agent additionally has two phosphorothioateinternucleotide linkages between the terminal three nucleotides at boththe 5′-end of the sense strand and at the 5′-end of the antisensestrand. In one embodiment, every nucleotide in the sense strand and theantisense strand of the RNAi agent, including the nucleotides that arepart of the motifs are modified nucleotides. In one embodiment eachresidue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g.,in an alternating motif. Optionally, the RNAi agent further comprises aligand (preferably GalNAc₃).

In one embodiment, the RNAi agent comprises sense and antisense strands,wherein the RNAi agent comprises a first strand having a length which isat least 25 and at most 29 nucleotides and a second strand having alength which is at most 30 nucleotides with at least one motif of three2′-O-methyl modifications on three consecutive nucleotides at position11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand andthe 5′ end of the second strand form a blunt end and the second strandis 1-4 nucleotides longer at its 3′ end than the first strand, whereinthe duplex region which is at least 25 nucleotides in length, and thesecond strand is sufficiently complementary to a target mRNA along atleast 19 nucleotide of the second strand length to reduce target geneexpression when the RNAi agent is introduced into a mammalian cell, andwherein dicer cleavage of the RNAi agent preferentially results in ansiRNA comprising the 3′ end of the second strand, thereby reducingexpression of the target gene in the mammal. Optionally, the RNAi agentfurther comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at leastone motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In one embodiment, the antisense strand of the RNAi agent can alsocontain at least one motif of three identical modifications on threeconsecutive nucleotides, where one of the motifs occurs at or near thecleavage site in the antisense strand

For an RNAi agent having a duplex region of 17-23 nucleotides in length,the cleavage site of the antisense strand is typically around the 10, 11and 12 positions from the 5′-end. Thus the motifs of three identicalmodifications may occur at the 9, 10, 11 positions; 10, 11, 12positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15positions of the antisense strand, the count starting from the 1^(st)nucleotide from the 5′-end of the antisense strand, or, the countstarting from the 1^(st) paired nucleotide within the duplex region fromthe 5′-end of the antisense strand. The cleavage site in the antisensestrand may also change according to the length of the duplex region ofthe RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesmay overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain morethan one motif of three identical modifications on three consecutivenucleotides. The first motif may occur at or near the cleavage site ofthe strand and the other motifs may be a wing modification. The term“wing modification” herein refers to a motif occurring at anotherportion of the strand that is separated from the motif at or near thecleavage site of the same strand. The wing modification is eitheradajacent to the first motif or is separated by at least one or morenucleotides. When the motifs are immediately adjacent to each other thenthe chemistry of the motifs are distinct from each other and when themotifs are separated by one or more nucleotide than the chemistries canbe the same or different. Two or more wing modifications may be present.For instance, when two wing modifications are present, each wingmodification may occur at one end relative to the first motif which isat or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the RNAi agent maycontain more than one motifs of three identical modifications on threeconsecutive nucleotides, with at least one of the motifs occurring at ornear the cleavage site of the strand. This antisense strand may alsocontain one or more wing modifications in an alignment similar to thewing modifications that may be present on the sense strand.

In one embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two terminal nucleotides at the 3′-end, 5′-end or both ends ofthe strand.

In another embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two paired nucleotides within the duplex region at the 3′-end,5′-end or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least one wing modification, the wing modifications may fallon the same end of the duplex region, and have an overlap of one, two orthree nucleotides.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least two wing modifications, the sense strand and theantisense strand can be so aligned that two modifications each from onestrand fall on one end of the duplex region, having an overlap of one,two or three nucleotides; two modifications each from one strand fall onthe other end of the duplex region, having an overlap of one, two orthree nucleotides; two modifications one strand fall on each side of thelead motif, having an overlap of one, two or three nucleotides in theduplex region.

In one embodiment, every nucleotide in the sense strand and antisensestrand of the RNAi agent, including the nucleotides that are part of themotifs, may be modified. Each nucleotide may be modified with the sameor different modification which can include one or more alteration ofone or both of the non-linking phosphate oxygens and/or of one or moreof the linking phosphate oxygens; alteration of a constituent of theribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesalereplacement of the phosphate moiety with “dephospho” linkers;modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking O of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of an RNA or may only occur in a single strand region of aRNA. For example, a phosphorothioate modification at a non-linking Oposition may only occur at one or both termini, may only occur in aterminal region, e.g., at a position on a terminal nucleotide or in thelast 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in doublestrand and single strand regions, particularly at termini. The 5′ end orends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, orin both. For example, it can be desirable to include purine nucleotidesin overhangs. In some embodiments all or some of the bases in a 3′ or 5′overhang may be modified, e.g., with a modification described herein.Modifications can include, e.g., the use of modifications at the 2′position of the ribose sugar with modifications that are known in theart, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or2′-O-methyl modified instead of the ribosugar of the nucleobase, andmodifications in the phosphate group, e.g., phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

In one embodiment, each residue of the sense strand and antisense strandis independently modified with LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-hydroxyl, or2′-fluoro. The strands can contain more than one modification. In oneembodiment, each residue of the sense strand and antisense strand isindependently modified with 2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of analternating pattern. The term “alternating motif” as used herein refersto a motif having one or more modifications, each modification occurringon alternating nucleotides of one strand. The alternating nucleotide mayrefer to one per every other nucleotide or one per every threenucleotides, or a similar pattern. For example, if A, B and C eachrepresent one type of modification to the nucleotide, the alternatingmotif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB. . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC. . . ,” etc.

The type of modifications contained in the alternating motif may be thesame or different. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In one embodiment, the RNAi agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′-3′ of the strand and the alternating motif in theantisense strand may start with “BABABA” from 5′-3′ of the strand withinthe duplex region. As another example, the alternating motif in thesense strand may start with “AABBAABB” from 5′-3′ of the strand and thealternating motif in the antisenese strand may start with “BBAABBAA”from 5′-3′ of the strand within the duplex region, so that there is acomplete or partial shift of the modification patterns between the sensestrand and the antisense strand.

In one embodiment, the RNAi agent comprises the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe sense strand initially has a shift relative to the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe antisense strand initially, i.e., the 2′-O-methyl modifiednucleotide on the sense strand base pairs with a 2′-F modifiednucleotide on the antisense strand and vice versa. The 1 position of thesense strand may start with the 2′-F modification, and the 1 position ofthe antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modificationson three consecutive nucleotides to the sense strand and/or antisensestrand interrupts the initial modification pattern present in the sensestrand and/or antisense strand. This interruption of the modificationpattern of the sense and/or antisense strand by introducing one or moremotifs of three identical modifications on three consecutive nucleotidesto the sense and/or antisense strand surprisingly enhances the genesilencing activity to the target gene.

In one embodiment, when the motif of three identical modifications onthree consecutive nucleotides is introduced to any of the strands, themodification of the nucleotide next to the motif is a differentmodification than the modification of the motif. For example, theportion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) .. . ,” where “Y” represents the modification of the motif of threeidentical modifications on three consecutive nucleotide, and “N_(a)” and“N_(b)” represent a modification to the nucleotide next to the motif“YYY” that is different than the modification of Y, and where N_(a) andN_(b) can be the same or different modifications. Alternatively, N_(a)and/or N_(b) may be present or absent when there is a wing modificationpresent.

The RNAi agent may further comprise at least one phosphorothioate ormethylphosphonate internucleotide linkage. The phosphorothioate ormethylphosphonate internucleotide linkage modification may occur on anynucleotide of the sense strand or antisense strand or both strands inany position of the strand. For instance, the internucleotide linkagemodification may occur on every nucleotide on the sense strand and/orantisense strand; each internucleotide linkage modification may occur inan alternating pattern on the sense strand and/or antisense strand; orthe sense strand or antisense strand may contain both internucleotidelinkage modifications in an alternating pattern. The alternating patternof the internucleotide linkage modification on the sense strand may bethe same or different from the antisense strand, and the alternatingpattern of the internucleotide linkage modification on the sense strandmay have a shift relative to the alternating pattern of theinternucleotide linkage modification on the antisense strand.

In one embodiment, the RNAi comprises a phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region may contain two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within the duplex region. For example, at least 2, 3, 4, orall the overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. These terminal three nucleotides may be at the 3′-end of theantisense strand, the 3′-end of the sense strand, the 5′-end of theantisense strand, and/or the 5′ end of the antisense strand.

In one embodiment, the 2 nucleotide overhang is at the 3′-end of theantisense strand, and there are two phosphorothioate internucleotidelinkages between the terminal three nucleotides, wherein two of thethree nucleotides are the overhang nucleotides, and the third nucleotideis a paired nucleotide next to the overhang nucleotide. Optionally, theRNAi agent may additionally have two phosphorothioate internucleotidelinkages between the terminal three nucleotides at both the 5′-end ofthe sense strand and at the 5′-end of the antisense strand.

In one embodiment, the RNAi agent comprises mismatch(es) with thetarget, within the duplex, or combinations thereof. A “mismatch” may benon-canonical base pairing or other than canonical pairing ofnucleotides. The mistmatch may occur in the overhang region or theduplex region. The base pair may be ranked on the basis of theirpropensity to promote dissociation or melting (e.g., on the free energyof association or dissociation of a particular pairing, the simplestapproach is to examine the pairs on an individual pair basis, thoughnext neighbor or similar analysis can also be used). In terms ofpromoting dissociation: A:U is preferred over G:C; G:U is preferred overG:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g.,non-canonical or other than canonical pairings (as described elsewhereherein) are preferred over canonical (A:T, A:U, G:C) pairings; andpairings which include a universal base are preferred over canonicalpairings. A “universal base” is a base that exhibits the ability toreplace any of the four normal bases (G, C, A, and U) withoutsignificantly destabilizing neighboring base-pair interactions ordisrupting the expected functional biochemical utility of the modifiedoligonucleotide. Non-limiting examples of universal bases include2′-deoxyinosine (hypoxanthine deoxynucleotide) or its derivatives,nitroazole analogues, and hydrophobic aromatic non-hydrogen-bondingbases.

In one embodiment, the RNAi agent comprises at least one of the first 1,2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end ofthe antisense strand independently selected from the group of: A:U, G:U,I:C, and mismatched pairs, e.g., non-canonical or other than canonicalpairings or pairings which include a universal base, to promote thedissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplexregion from the 5′-end in the antisense strand is selected from thegroup consisting of A, dA, dU, U, and dT. Alternatively, at least one ofthe first 1, 2 or 3 base pair within the duplex region from the 5′-endof the antisense strand is an AU base pair. For example, the first basepair within the duplex region from the 5′-end of the antisense strand isan AU base pair.

In another embodiment, the nucleotide at the 3′-end of the sense strandis deoxy-thymine (dT). In another embodiment, the nucleotide at the3′-end of the antisense strand is deoxy-thymine (dT). In one embodiment,there is a short sequence of deoxy-thymine nucleotides, for example, twodT nucleotides on the 3′-end of the sense and/or antisense strand.

In one embodiment, the sense strand sequence may be represented byformula (I):

(I) 5′n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. Preferably YYYis all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications ofalternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand. For example, when the RNAi agent has a duplex regionof 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7,8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of—the sensestrand, the count starting from the 1^(st) nucleotide, from the 5′-end;or optionally, the count starting at the 1^(st) paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:

(Ib) 5′ n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′; (Ic) 5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q) 3′;  or (Id) 5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′.

When the sense strand is represented by formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2 or 0 modified nucleotides. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Id), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1,2, 3, 4, 5 or 6 Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may berepresented by the formula:

(Ia) 5′ n_(p)-N_(a)-YYY-N_(a)-n_(q) 3′.

When the sense strand is represented by formula (Ia), each N_(a)independently can represent an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may berepresented by formula (II):

(II) 5′n_(q′)-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)_(l)-N_(a)′-n_(p)′ 3′

wherein:

k and 1 are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification;

and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the RNAi agent has a duplex region of 17-23nucleotidein length, the Y′Y′Y′ motif can occur at positions 9, 10, 11;10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisensestrand, with the count starting from the 1^(st) nucleotide, from the5′-end; or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end. Preferably, theY′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and 1 is 0, or k is 0 and 1 is 1, or both kand 1 are 1.

The antisense strand can therefore be represented by the followingformulas:

(IIb) 5′ n_(q′)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n^(p′) 3′; (IIc) 5′n_(q′)-N_(a)′-Y′Y′Y′-N_(b)′-X′X′X′-n_(p′) 3′; or (IId) 5′n_(q′)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′-X′X′X′-N^(a)′-n^(p′) 3′.

When the antisense strand is represented by formula (IIb), N_(b)represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5 or 6.

In other embodiments, k is 0 and 1 is 0 and the antisense strand may berepresented by the formula:

(Ia) 5′ n_(p′)-N_(a′)-Y′Y′Y′-N_(a′)-n_(q′) 3′.

When the antisense strand is represented as formula (IIa), each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. Forexample, each nucleotide of the sense strand and antisense strand isindependently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′,Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYYmotif occurring at 9, 10 and 11 positions of the strand when the duplexregion is 21 nt, the count starting from the 1^(st) nucleotide from the5′-end, or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification. The sense strand may additionally contain XXX motifor ZZZ motifs as wing modifications at the opposite end of the duplexregion; and XXX and ZZZ each independently represents a 2′-OMemodification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe 1^(st) nucleotide from the 5′-end, or optionally, the count startingat the 1^(st) paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification.

The sense strand represented by any one of the above formulas (Ia),(Ib), (Ic), and (Id) forms a duplex with a antisense strand beingrepresented by any one of formulas (IIa), (IIb), (IIc), and (IId),respectively.

Accordingly, the RNAi agents for use in the methods of the invention maycomprise a sense strand and an antisense strand, each strand having 14to 30 nucleotides, the RNAi duplex represented by formula (III):

(III) sense: 5′n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b) independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein

each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or may not bepresent, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0and j is 1; or both i and j are 0; or both i and j are 1. In anotherembodiment, k is 0 and 1 is 0; or k is 1 and 1 is 0; k is 0 and 1 is 1;or both k and 1 are 0; or both k and 1 are 1.

Exemplary combinations of the sense strand and antisense strand forminga RNAi duplex include the formulas below:

(IIIa) 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′ 3′n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′n_(q)′ 5′ (IIIb) 5′n_(p)-N_(a)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ 3′n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′n_(q)′ 5′ (IIIc) 5′n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(a)-n_(q) 3′ 3′n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-Na′-n_(q)′ 5′ (IIId) 5′n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ 3′n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)-n_(q)′ 5′

When the RNAi agent is represented by formula (IIIa), each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a),N_(a) independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b)and N_(b) independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId)may be the same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), at least one of the Y nucleotides may form a basepair with one of the Y′ nucleotides. Alternatively, at least two of theY nucleotides form base pairs with the corresponding Y′ nucleotides; orall three of the Y nucleotides all form base pairs with thecorresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at leastone of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIc) or (IIId), at leastone of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is differentthan the modification on the Y′ nucleotide, the modification on the Znucleotide is different than the modification on the Z′ nucleotide,and/or the modification on the X nucleotide is different than themodification on the X′ nucleotide.

In one embodiment, when the RNAi agent is represented by formula (IIId),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. Inanother embodiment, when the RNAi agent is represented by formula(IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications and n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide a via phosphorothioate linkage. In yet anotherembodiment, when the RNAi agent is represented by formula (IIId), theN_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0and at least one n_(p)′ is linked to a neighboring nucleotide viaphosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker. In another embodiment, when the RNAi agent isrepresented by formula (IIId), the N_(a) modifications are 2′-O-methylor 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linkedto a neighboring nucleotide via phosphorothioate linkage, the sensestrand comprises at least one phosphorothioate linkage, and the sensestrand is conjugated to one or more GalNAc derivatives attached througha bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications,n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia phosphorothioate linkage, the sense strand comprises at least onephosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker.

In one embodiment, the RNAi agent is a multimer containing at least twoduplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and(IIId), wherein the duplexes are connected by a linker. The linker canbe cleavable or non-cleavable. Optionally, the multimer furthercomprises a ligand. Each of the duplexes can target the same gene or twodifferent genes; or each of the duplexes can target same gene at twodifferent target sites.

In one embodiment, the RNAi agent is a multimer containing three, four,five, six or more duplexes represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), wherein the duplexes are connected by a linker. Thelinker can be cleavable or non-cleavable. Optionally, the multimerfurther comprises a ligand. Each of the duplexes can target the samegene or two different genes; or each of the duplexes can target samegene at two different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa),(IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, andone or both of the 3′ ends and are optionally conjugated to a ligand.Each of the agents can target the same gene or two different genes; oreach of the agents can target same gene at two different target sites.

Various publications describe multimeric RNAi agents that can be used inthe methods of the invention. Such publications include WO2007/091269,U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 andWO2011/031520 the entire contents of each of which are herebyincorporated herein by reference.

The RNAi agent that contains conjugations of one or more carbohydratemoieties to a RNAi agent can optimize one or more properties of the RNAiagent. In many cases, the carbohydrate moiety will be attached to amodified subunit of the RNAi agent. For example, the ribose sugar of oneor more ribonucleotide subunits of a dsRNA agent can be replaced withanother moiety, e.g., a non-carbohydrate (preferably cyclic) carrier towhich is attached a carbohydrate ligand. A ribonucleotide subunit inwhich the ribose sugar of the subunit has been so replaced is referredto herein as a ribose replacement modification subunit (RRMS). A cycliccarrier may be a carbocyclic ring system, i.e., all ring atoms arecarbon atoms, or a heterocyclic ring system, i.e., one or more ringatoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cycliccarrier may be a monocyclic ring system, or may contain two or morerings, e.g. fused rings. The cyclic carrier may be a fully saturatedring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein thecarrier can be cyclic group or acyclic group; preferably, the cyclicgroup is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl anddecalin; preferably, the acyclic group is selected from serinol backboneor diethanolamine backbone.

In certain specific embodiments, the RNAi agent for use in the methodsof the invention is an agent selected from the group of agents listed inany one of Tables 1, 2, 5, and 7.

These agents may further comprise a ligand.

A. Ligands

The double-stranded RNA (dsRNA) agents of the invention may optionallybe conjugated to one or more ligands. The ligand can be attached to thesense strand, antisense strand or both strands, at the 3′-end, 5′-end orboth ends. For instance, the ligand may be conjugated to the sensestrand. In preferred embodiments, the ligand is conjugated to the 3′-endof the sense strand. In one preferred embodiment, the ligand is a GalNAcligand. In particularly preferred embodiments, the ligand is GalNAc₃:

In some embodiments, the ligand, e.g., GalNAc ligand, is attached to the3′ end of the RNAi agent. In one embodiment, the RNAi agent isconjugated to the ligand, e.g., GalNAc ligand, as shown in the followingschematic

wherein X is O or S. In one embodiment, X is O.

A wide variety of entities can be coupled to the RNAi agents of thepresent invention. Preferred moieties are ligands, which are coupled,preferably covalently, either directly or indirectly via an interveningtether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of the molecule into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, receptor e.g., acellular or organ compartment, tissue, organ or region of the body, as,e.g., compared to a species absent such a ligand. Ligands providingenhanced affinity for a selected target are also termed targetingligands.

Some ligands can have endosomolytic properties. The endosomolyticligands promote the lysis of the endosome and/or transport of thecomposition of the invention, or its components, from the endosome tothe cytoplasm of the cell. The endosomolytic ligand may be a polyanionicpeptide or peptidomimetic which shows pH-dependent membrane activity andfusogenicity. In one embodiment, the endosomolytic ligand assumes itsactive conformation at endosomal pH. The “active” conformation is thatconformation in which the endosomolytic ligand promotes lysis of theendosome and/or transport of the composition of the invention, or itscomponents, from the endosome to the cytoplasm of the cell. Exemplaryendosomolytic ligands include the GALA peptide (Subbarao et al.,Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al., J.Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk etal., Biochem. Biophys. Acta, 2002, 1559: 56-68). In one embodiment, theendosomolytic component may contain a chemical group (e.g., an aminoacid) which will undergo a change in charge or protonation in responseto a change in pH. The endosomolytic component may be linear orbranched.

Ligands can improve transport, hybridization, and specificity propertiesand may also improve nuclease resistance of the resultant natural ormodified oligoribonucleotide, or a polymeric molecule comprising anycombination of monomers described herein and/or natural or modifiedribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; and nuclease-resistanceconferring moieties. General examples include lipids, steroids,vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL),high-density lipoprotein (HDL), or globulin); a carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronicacid); or a lipid. The ligand may also be a recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid,an oligonucleotide (e.g., an aptamer). Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer.

Other examples of ligands include dyes, intercalating agents (e.g.,acridines), cross-linkers (e.g., psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases or a chelator(e.g., EDTA), lipophilic molecules, e.g., cholesterol, cholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g.,biotin), transport/absorption facilitators (e.g., aspirin, vitamin E,folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose, oraptamers. The ligand can be, for example, a lipopolysaccharide, anactivator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the oligonucleotide into the cellby, for example, activating an inflammatory response. Exemplary ligandsthat would have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, naproxen or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include B vitamins, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HAS, low density lipoprotein (LDL) andhigh-density lipoprotein (HDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long. A peptide or peptidomimetic can be, for example, a cellpermeation peptide, cationic peptide, amphipathic peptide, orhydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). Thepeptide moiety can be a dendrimer peptide, constrained peptide orcrosslinked peptide. In another alternative, the peptide moiety caninclude a hydrophobic membrane translocation sequence (MTS). Anexemplary hydrophobic MTS-containing peptide is RFGF having the aminoacid sequence AAVALLPAVLLALLAP (SEQ ID NO:29). An RFGF analogue (e.g.,amino acid sequence AALLPVLLAAP (SEQ ID NO:30)) containing a hydrophobicMTS can also be a targeting moiety. The peptide moiety can be a“delivery” peptide, which can carry large polar molecules includingpeptides, oligonucleotides, and protein across cell membranes. Forexample, sequences from the HIV Tat protein (GRKKRRQRRRPPQ; SEQ IDNO:31) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK; SEQ IDNO:32) have been found to be capable of functioning as deliverypeptides. A peptide or peptidomimetic can be encoded by a randomsequence of DNA, such as a peptide identified from a phage-displaylibrary, or one-bead-one-compound (OBOC) combinatorial library (Lam etal., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetictethered to an iRNA agent via an incorporated monomer unit is a celltargeting peptide such as an arginine-glycine-aspartic acid(RGD)-peptide, or RGD mimic. A peptide moiety can range in length fromabout 5 amino acids to about 40 amino acids. The peptide moieties canhave a structural modification, such as to increase stability or directconformational properties. Any of the structural modifications describedbelow can be utilized. An RGD peptide moiety can be used to target atumor cell, such as an endothelial tumor cell or a breast cancer tumorcell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptidecan facilitate targeting of an iRNA agent to tumors of a variety ofother tissues, including the lung, kidney, spleen, or liver (Aoki etal., Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD peptidewill facilitate targeting of an iRNA agent to the kidney. The RGDpeptide can be linear or cyclic, and can be modified, e.g., glycosylatedor methylated to facilitate targeting to specific tissues. For example,a glycosylated RGD peptide can deliver an iRNA agent to a tumor cellexpressing α_(v)β₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).Peptides that target markers enriched in proliferating cells can beused. For example, RGD containing peptides and peptidomimetics cantarget cancer cells, in particular cells that exhibit an integrin. Thus,one could use RGD peptides, cyclic peptides containing RGD, RGD peptidesthat include D-amino acids, as well as synthetic RGD mimics. In additionto RGD, one can use other moieties that target the integrin ligand.Generally, such ligands can be used to control proliferating cells andangiogeneis. Preferred conjugates of this type of ligand target PECAM-1,VEGF, or other cancer gene, e.g., a cancer gene described herein.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide can be an amphipathic α-helicalpeptide. Exemplary amphipathic α-helical peptides include, but are notlimited to, cecropins, lycotoxins, paradaxins, buforin, CPF,bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clavapeptides, hagfish intestinal antimicrobial peptides (HFIAPs),magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂Apeptides, Xenopus peptides, esculentinis-1, and caerins. A number offactors will preferably be considered to maintain the integrity of helixstability. For example, a maximum number of helix stabilization residueswill be utilized (e.g., leu, ala, or lys), and a minimum number helixdestabilization residues will be utilized (e.g., proline, or cyclicmonomeric units. The capping residue will be considered (for example Glyis an exemplary N-capping residue and/or C-terminal amidation can beused to provide an extra H-bond to stabilize the helix. Formation ofsalt bridges between residues with opposite charges, separated by i±3,or i±4 positions can provide stability. For example, cationic residuessuch as lysine, arginine, homo-arginine, ornithine or histidine can formsalt bridges with the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting aspecific receptor. Examples are: folate, GalNAc, galactose, mannose,mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster,galactose cluster, or an apatamer. A cluster is a combination of two ormore sugar units. The targeting ligands also include integrin receptorligands, Chemokine receptor ligands, transferrin, biotin, serotoninreceptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDLligands. The ligands can also be based on nucleic acid, e.g., anaptamer. The aptamer can be unmodified or have any combination ofmodifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles,PEIs, peptides, fusogenic peptides, polycaboxylates, polyacations,masked oligo or poly cations or anions, acetals, polyacetals,ketals/polyketyals, orthoesters, polymers with masked or unmaskedcationic or anionic charges, dendrimers with masked or unmasked cationicor anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulators includelipophiles, bile acids, steroids, phospholipid analogues, peptides,protein binding agents, PEG, vitamins etc. Examplary PK modulatorsinclude, but are not limited to, cholesterol, fatty acids, cholic acid,lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids,sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.,oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,comprising multiple phosphorothioate linkages in the backbaone are alsoamenable to the present invention as ligands (e.g., as PK modulatingligands).

In addition, aptamers that bind serum components (e.g., serum proteins)are also amenable to the present invention as PK modulating ligands.

Other ligand conjugates amenable to the invention are described in U.S.patent application Ser. No. 10/916,185, filed Aug. 10, 2004; Ser. No.10/946,873, filed Sep. 21, 2004; Ser. No. 10/833,934, filed Aug. 3,2007; Ser. No. 11/115,989 filed Apr. 27, 2005 and Ser. No. 11/944,227filed Nov. 21, 2007, which are incorporated by reference in theirentireties for all purposes.

When two or more ligands are present, the ligands can all have sameproperties, all have different properties or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

Ligands can be coupled to the oligonucleotides at various places, forexample, 3′-end, 5′-end, and/or at an internal position. In preferredembodiments, the ligand is attached to the oligonucleotides via anintervening tether, e.g., a carrier described herein. The ligand ortethered ligand may be present on a monomer when the monomer isincorporated into the growing strand. In some embodiments, the ligandmay be incorporated via coupling to a “precursor” monomer after the“precursor” monomer has been incorporated into the growing strand. Forexample, a monomer having, e.g., an amino-terminated tether (i.e.,having no associated ligand), e.g., TAP-(CH₂)_(n)NH₂ may be incorporatedinto a growing oligonucelotide strand. In a subsequent operation, i.e.,after incorporation of the precursor monomer into the strand, a ligandhaving an electrophilic group, e.g., a pentafluorophenyl ester oraldehyde group, can subsequently be attached to the precursor monomer bycoupling the electrophilic group of the ligand with the terminalnucleophilic group of the precursor monomer's tether.

In another example, a monomer having a chemical group suitable fortaking part in Click Chemistry reaction may be incorporated, e.g., anazide or alkyne terminated tether/linker. In a subsequent operation,i.e., after incorporation of the precursor monomer into the strand, aligand having complementary chemical group, e.g. an alkyne or azide canbe attached to the precursor monomer by coupling the alkyne and theazide together.

For double-stranded oligonucleotides, ligands can be attached to one orboth strands. In some embodiments, a double-stranded iRNA agent containsa ligand conjugated to the sense strand. In other embodiments, adouble-stranded iRNA agent contains a ligand conjugated to the antisensestrand.

In some embodiments, ligand can be conjugated to nucleobases, sugarmoieties, or internucleosidic linkages of nucleic acid molecules.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a conjugate moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a conjugate moiety. Conjugation to sugar moieties ofnucleosides can occur at any carbon atom. Example carbon atoms of asugar moiety that can be attached to a conjugate moiety include the 2′,3′, and 5′ carbon atoms. The 1′ position can also be attached to aconjugate moiety, such as in an abasic residue. Internucleosidiclinkages can also bear conjugate moieties. For phosphorus-containinglinkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate,phosphoroamidate, and the like), the conjugate moiety can be attacheddirectly to the phosphorus atom or to an O, N, or S atom bound to thephosphorus atom. For amine- or amide-containing internucleosidiclinkages (e.g., PNA), the conjugate moiety can be attached to thenitrogen atom of the amine or amide or to an adjacent carbon atom.

Any suitable ligand in the field of RNA interference may be used,although the ligand is typically a carbohydrate e.g. monosaccharide(such as GalNAc), disaccharide, trisaccharide, tetrasaccharide,polysaccharide.

Linkers that conjugate the ligand to the nucleic acid include thosediscussed above. For example, the ligand can be one or more GalNAc(N-acetylglucosamine) derivatives attached through a bivalent ortrivalent branched linker.

In one embodiment, the dsRNA of the invention is conjugated to abivalent and trivalent branched linkers include the structures shown inany of formula (IV)-(VII):

wherein:

q^(2A), q^(2B), q^(3A), q^(3B), q4^(A), q^(4B), q^(5A), q^(5B) andq^(5C) represent independently for each occurrence 0-20 and wherein therepeating unit can be the same or different;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and

R^(a) is H or amino acid side chain.

Trivalent conjugating GalNAc derivatives are particularly useful for usewith RNAi agents for inhibiting the expression of a target gene, such asthose of formula (VII):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such asGalNAc derivative. Examples of suitable bivalent and trivalent branchedlinker groups conjugating GalNAc derivatives include, but are notlimited to, the following compounds:

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of thisinvention, are iRNA compounds, preferably dsRNAs, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAstypically contain at least one region wherein the RNA is modified so asto confer upon the iRNA increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the iRNA can serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof iRNA inhibition of gene expression. Consequently, comparable resultscan often be obtained with shorter iRNAs when chimeric dsRNAs are used,compared to phosphorothioate deoxy dsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction can be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

In some embodiments, method double-stranded RNAi agent of the inventionis selected from the group consisting of AD-58681, AD-59054, AD-61719,and AD-61444.

III. Delivery of an iRNA of the Invention

The delivery of an iRNA agent of the invention to a cell e.g., a cellwithin a subject, such as a human subject (e.g., a subject in needthereof, such as a subject having a Serpina1 deficiency-associateddisorder, e.g., a Serpina1 deficiency liver disorder) can be achieved ina number of different ways. For example, delivery may be performed bycontacting a cell with an iRNA of the invention either in vitro or invivo. In vivo delivery may also be performed directly by administering acomposition comprising an iRNA, e.g., a dsRNA, to a subject.Alternatively, in vivo delivery may be performed indirectly byadministering one or more vectors that encode and direct the expressionof the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. Thenon-specific effects of an iRNA can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J., et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274)and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006)Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNAinterference has also shown success with local delivery to the CNS bydirect injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMCNeurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528;Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects. iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., etal (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer hasbeen shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O., et al (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H., et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328;Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet Me., et al (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., etal (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA formsa complex with cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the Serpina1 gene can be expressed from transcriptionunits inserted into DNA or RNA vectors (see, e.g., Couture, A, et al.,TIG. (1996), 12:5-10; Skillern, A., et al., International PCTPublication No. WO 00/22113, Conrad, International PCT Publication No.WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can betransient (on the order of hours to weeks) or sustained (weeks to monthsor longer), depending upon the specific construct used and the targettissue or cell type. These transgenes can be introduced as a linearconstruct, a circular plasmid, or a viral vector, which can be anintegrating or non-integrating vector. The transgene can also beconstructed to permit it to be inherited as an extrachromosomal plasmid(Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as acomplex with cationic lipid carriers (e.g., Oligofectamine) ornon-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipidtransfections for iRNA-mediated knockdowns targeting different regionsof a target RNA over a period of a week or more are also contemplated bythe invention. Successful introduction of vectors into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofcells ex vivo can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of an iRNA will include regulatoryelements (promoter, enhancer, etc.) sufficient for expression of theiRNA in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the iRNA can be precisely regulated, for example, by usingan inducible regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of dsRNA expression in cells or inmammals include, for example, regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the iRNA transgene.

Viral vectors that contain nucleic acid sequences encoding an iRNA canbe used. For example, a retroviral vector can be used (see Miller etal., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectorscontain the components necessary for the correct packaging of the viralgenome and integration into the host cell DNA. The nucleic acidsequences encoding an iRNA are cloned into one or more vectors, whichfacilitate delivery of the nucleic acid into a patient. More detailabout retroviral vectors can be found, for example, in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993). Lentiviral vectors contemplated for use include, forexample, the HIV based vectors described in U.S. Pat. Nos. 6,143,520;5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs of theinvention. Adenoviruses are especially attractive vehicles, e.g., fordelivering genes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitableAV vector for expressing an iRNA featured in the invention, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Adeno-associated virus (AAV) vectors may also be used to delivery aniRNA of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med.204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, theiRNA can be expressed as two separate, complementary single-stranded RNAmolecules from a recombinant AAV vector having, for example, either theU6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. SuitableAAV vectors for expressing the dsRNA featured in the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent ApplicationNo. WO 94/13788; and International Patent Application No. WO 93/24641,the entire disclosures of which are herein incorporated by reference.

Another viral vector suitable for delivery of an iRNA of the inevtion isa pox virus such as a vaccinia virus, for example an attenuated vacciniasuch as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl poxor canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Forexample, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes; see, e.g.,Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosureof which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

III. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful fortreating a disease or disorder associated with the expression oractivity of a Serpina1 gene, e.g., a Serpina1 deficiency-associateddisorder, e.g., a Serpina1 deficiency liver disorder. Suchpharmaceutical compositions are formulated based on the mode ofdelivery. One example is compositions that are formulated for systemicadministration via parenteral delivery, e.g., by intravenous (IV)delivery. Another example is compositions that are formulated for directdelivery into the brain parenchyma, e.g., by infusion into the brain,such as by continuous pump infusion.

The pharmaceutical compositions comprising RNAi agents of the inventionmay be, for example, solutions with or without a buffer, or compositionscontaining pharmaceutically acceptable carriers. Such compositionsinclude, for example, aqueous or crystalline compositions, liposomalformulations, micellar formulations, emulsions, and gene therapyvectors.

In the methods of the invention, the RNAi agent may be administered in asolution. A free RNAi agent may be administered in an unbufferedsolution, e.g., in saline or in water. Alternatively, the free siRNA mayalso be administered in a suitable buffer solution. The buffer solutionmay comprise acetate, citrate, prolamine, carbonate, or phosphate, orany combination thereof. In a preferred embodiment, the buffer solutionis phosphate buffered saline (PBS). The pH and osmolarity of the buffersolution containing the RNAi agent can be adjusted such that it issuitable for administering to a subject.

In some embodiments, the buffer solution further comprises an agent forcontrolling the osmolarity of the solution, such that the osmolarity iskept at a desired value, e.g., at the physiologic values of the humanplasma. Solutes which can be added to the buffer solution to control theosmolarity include, but are not limited to, proteins, peptides, aminoacids, non-metabolized polymers, vitamins, ions, sugars, metabolites,organic acids, lipids, or salts. In some embodiments, the agent forcontrolling the osmolarity of the solution is a salt. In certainembodiments, the agent for controlling the osmolarity of the solution issodium chloride or potassium chloride.

The pharmaceutical compositions of the invention may be administered indosages sufficient to inhibit expression of a Serpina1 gene. In general,a suitable dose of an iRNA of the invention will be in the range ofabout 0.001 to about 200.0 milligrams per kilogram body weight of therecipient per day, generally in the range of about 1 to 50 mg perkilogram body weight per day. For example, the dsRNA can be administeredat about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg,about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 10 mg/kg, about 20mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg per singledose.

For example, the RNAi agent, e.g., dsRNA, may be administered at a doseof about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg.Values and ranges intermediate to the recited values are also intendedto be part of this invention.

In another embodiment, the RNAi agent, e.g., dsRNA, is administered at adose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

For example, the RNAi agent, e.g., dsRNA, may be administered at a doseof about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In another embodiment, the RNAi agent, e.g., dsRNA, is administered at adose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about1 to about 50 mg/mg, about 1.5 to about 50 mg/kg, about 2 to about 50mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40to about 45 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35to about 40 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment, thedsRNA is administered at a dose of about 10 mg/kg to about 30 mg/kg.Values and ranges intermediate to the recited values are also intendedto be part of this invention.

For example, subjects can be administered a therapeutic amount of iRNA,such as about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5,13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5,20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5,27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

In certain embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and a lipid, subjects can beadministered a therapeutic amount of iRNA, such as about 0.01 mg/kg toabout 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg toabout 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg toabout 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg toabout 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg toabout 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg toabout 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg toabout 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about 2.5mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg,about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention. For example, the dsRNA may be administered at adose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

In certain embodiments of the invention, for example, when adouble-stranded RNAi agent includes modifications (e.g., one or moremotifs of three identical modifications on three consecutivenucleotides, including one such motif at or near the cleavage site ofthe agent), six phosphorothioate linkages, and a ligand, such an agentis administered at a dose of about 0.01 to about 0.5 mg/kg, about 0.01to about 0.4 mg/kg, about 0.01 to about 0.3 mg/kg, about 0.01 to about0.2 mg/kg, about 0.01 to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09mg/kg, about 0.01 mg/kg to about 0.08 mg/kg, about 0.01 mg/kg to about0.07 mg/kg, about 0.01 mg/kg to about 0.06 mg/kg, about 0.01 mg/kg toabout 0.05 mg/kg, about 0.02 to about 0.5 mg/kg, about 0.02 to about 0.4mg/kg, about 0.02 to about 0.3 mg/kg, about 0.02 to about 0.2 mg/kg,about 0.02 to about 0.1 mg/kg, about 0.02 mg/kg to about 0.09 mg/kg,about 0.02 mg/kg to about 0.08 mg/kg, about 0.02 mg/kg to about 0.07mg/kg, about 0.02 mg/kg to about 0.06 mg/kg, about 0.02 mg/kg to about0.05 mg/kg, about 0.03 to about 0.5 mg/kg, about 0.03 to about 0.4mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg,about 0.03 to about 0.1 mg/kg, about 0.03 mg/kg to about 0.09 mg/kg,about 0.03 mg/kg to about 0.08 mg/kg, about 0.03 mg/kg to about 0.07mg/kg, about 0.03 mg/kg to about 0.06 mg/kg, about 0.03 mg/kg to about0.05 mg/kg, about 0.04 to about 0.5 mg/kg, about 0.04 to about 0.4mg/kg, about 0.04 to about 0.3 mg/kg, about 0.04 to about 0.2 mg/kg,about 0.04 to about 0.1 mg/kg, about 0.04 mg/kg to about 0.09 mg/kg,about 0.04 mg/kg to about 0.08 mg/kg, about 0.04 mg/kg to about 0.07mg/kg, about 0.04 mg/kg to about 0.06 mg/kg, about 0.05 to about 0.5mg/kg, about 0.05 to about 0.4 mg/kg, about 0.05 to about 0.3 mg/kg,about 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.05mg/kg to about 0.09 mg/kg, about 0.05 mg/kg to about 0.08 mg/kg, orabout 0.05 mg/kg to about 0.07 mg/kg. Values and ranges intermediate tothe foregoing recited values are also intended to be part of thisinvention, e.g., the RNAi agent may be administered to the subject at adose of about 0.015 mg/kg to about 0.45 mg/mg.

For example, the RNAi agent, e.g., RNAi agent in a pharmaceuticalcomposition, may be administered at a dose of about 0.01 mg/kg, 0.0125mg/kg, 0.015 mg/kg, 0.0175 mg/kg, 0.02 mg/kg, 0.0225 mg/kg, 0.025 mg/kg,0.0275 mg/kg, 0.03 mg/kg, 0.0325 mg/kg, 0.035 mg/kg, 0.0375 mg/kg, 0.04mg/kg, 0.0425 mg/kg, 0.045 mg/kg, 0.0475 mg/kg, 0.05 mg/kg, 0.0525mg/kg, 0.055 mg/kg, 0.0575 mg/kg, 0.06 mg/kg, 0.0625 mg/kg, 0.065 mg/kg,0.0675 mg/kg, 0.07 mg/kg, 0.0725 mg/kg, 0.075 mg/kg, 0.0775 mg/kg, 0.08mg/kg, 0.0825 mg/kg, 0.085 mg/kg, 0.0875 mg/kg, 0.09 mg/kg, 0.0925mg/kg, 0.095 mg/kg, 0.0975 mg/kg, 0.1 mg/kg, 0.125 mg/kg, 0.15 mg/kg,0.175 mg/kg, 0.2 mg/kg, 0.225 mg/kg, 0.25 mg/kg, 0.275 mg/kg, 0.3 mg/kg,0.325 mg/kg, 0.35 mg/kg, 0.375 mg/kg, 0.4 mg/kg, 0.425 mg/kg, 0.45mg/kg, 0.475 mg/kg, or about 0.5 mg/kg. Values intermediate to theforegoing recited values are also intended to be part of this invention.

The pharmaceutical composition can be administered once daily, or theiRNA can be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, theiRNA contained in each sub-dose must be correspondingly smaller in orderto achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of theiRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4week intervals. In some embodiments of the invention, a single dose ofthe pharmaceutical compositions of the invention is administered onceper week. In other embodiments of the invention, a single dose of thepharmaceutical compositions of the invention is administered bi-monthly.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as a liver disorder that wouldbenefit from reduction in the expression of Serpina1. Such models can beused for in vivo testing of iRNA, as well as for determining atherapeutically effective dose. Suitable mouse models are known in theart and include, for example, a mouse containing a transgene expressinghuman Serpina1.

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration. The iRNA can be delivered in a manner to target aparticular tissue, such as the liver (e.g., the hepatocytes of theliver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention can be encapsulated within liposomes or can form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs canbe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof). Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA composition. The lipophilic material isolates theaqueous interior from an aqueous exterior, which typically does notinclude the iRNA composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the iRNA are delivered into the cell where theiRNA can specifically bind to a target RNA and can mediate RNAi. In somecases the liposomes are also specifically targeted, e.g., to direct theiRNA to particular cell types.

A liposome containing a RNAi agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAiagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the RNAi agentand condense around the RNAi agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of RNAi agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad.Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No.5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al.Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci.75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984;Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging RNAi agent preparations intoliposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH-sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al., Journal of Controlled Release, 1992, 19,269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550,1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human GeneTher. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J.11:417, 1992.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside GM1, galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver RNAi agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated RNAi agents in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of RNAi agent (see, e.g.,Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer RNAi agent into the skin. In some implementations,liposomes are used for delivering RNAi agent to epidermal cells and alsoto enhance the penetration of RNAi agent into dermal tissues, e.g., intoskin. For example, the liposomes can be applied topically. Topicaldelivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992,vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992,259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690,1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth.Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth.Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad.Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith RNAi agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include RNAi agentcan be delivered, for example, subcutaneously by infection in order todeliver RNAi agent to keratinocytes in the skin. In order to crossintact mammalian skin, lipid vesicles must pass through a series of finepores, each with a diameter less than 50 nm, under the influence of asuitable transdermal gradient. In addition, due to the lipid properties,these transferosomes can be self-optimizing (adaptive to the shape ofpores, e.g., in the skin), self-repairing, and can frequently reachtheir targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided asmicellar formulations. “Micelles” are defined herein as a particulartype of molecular assembly in which amphipathic molecules are arrangedin a spherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in alipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle. LNPs contain a cationic lipid, a non-cationic lipid, and alipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). LNPs are extremely useful for systemic applications, as theyexhibit extended circulation lifetimes following intravenous (i.v.)injection and accumulate at distal sites (e.g., sites physicallyseparated from the administration site). LNPs include “pSPLP,” whichinclude an encapsulated condensing agent-nucleic acid complex as setforth in PCT Publication No. WO 00/03683. The particles of the presentinvention typically have a mean diameter of about 50 nm to about 150 nm,more typically about 60 nm to about 130 nm, more typically about 70 nmto about 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present invention are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S.Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. Ranges intermediate to the above recited ranges are alsocontemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN 100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech GI), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference. In one embodiment, thelipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HC1 (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which isincorporated herein by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are described in Table A.

TABLE A cationic lipid/non-cationic lipid/cholesterol/PEG-lipidconjugate Ionizable/Cationic Lipid Lipid:siRNA ratio LNP-11,2-Dilinolenyloxy-N,N-dimethylaminopropaneDLinDMA/DPPC/Cholesterol/PEG-cDMA (DLinDMA) (57.1/7.1/34.4/1.4)lipid:siRNA ~7:1 2-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DPPC/Cholesterol/PEG-cDMA dioxolane (XTC) 57.1/7.1/34.4/1.4lipid:siRNA ~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5lipid:siRNA ~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5lipid:siRNA ~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA10:1 LNP10 (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-ALN100/DSPC/Cholesterol/PEG-DMG octadeca-9,12-dienyl)tetrahydro-3aH-50/10/38.5/1.5 cyclopenta[d][1,3]dioxol-5-amine (ALN100) Lipid:siRNA10:1 LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-MC-3/DSPC/Cholesterol/PEG-DMG tetraen-19-yl 4-(dimethylamino)butanoate50/10/38.5/1.5 (MC3) Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2-Tech G1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:siRNA 10:1yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1

DSPC: distearoylphosphatidylcholine

DPPC: dipalmitoylphosphatidylcholine

PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000)

PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg molwt of 2000)

PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg molwt of 2000)

LNP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No.61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun.10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009;U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, andInternational Application No. PCT/US2010/022614, filed Jan. 29, 2010,which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. Publication No.2010/0324120, filed Jun. 10, 2010, the entire contents of which arehereby incorporated by reference. ALNY-100 comprising formulations aredescribed, e.g., International patent application number PCT/US09/63933,filed on Nov. 10, 2009, which is hereby incorporated by reference.C12-200 comprising formulations are described in U.S. Provisional Ser.No. 61/175,770, filed May 5, 2009 and International Application No.PCT/US10/33777, filed May 5, 2010, which are hereby incorporated byreference.

Synthesis of Ionizable/Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in thenucleic acid-lipid particles of the invention can be prepared by knownorganic synthesis techniques, including the methods described in moredetail in the Examples. All substituents are as defined below unlessindicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms can be optionally oxidized, and the nitrogenheteroatom can be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle can be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy,—SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same ordifferent and independently hydrogen, alkyl or heterocycle, and each ofsaid alkyl and heterocycle substituents can be further substituted withone or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy,—NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and—SOnNRxRy.

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the invention can require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, Protective Groups in OrganicSynthesis, Green, T. W. et al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles of the invention areformulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring. In some embodiments, the cationic lipid is XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, thelipid of formula A above can be made by the following Reaction Schemes 1or 2, wherein all substituents are as defined above unless indicatedotherwise.

Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl,each can be optionally substituted, and R3 and R4 are independentlylower alkyl or R3 and R4 can be taken together to form an optionallysubstituted heterocyclic ring, can be prepared according to Scheme 1.Ketone 1 and bromide 2 can be purchased or prepared according to methodsknown to those of ordinary skill in the art. Reaction of 1 and 2 yieldsketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.The lipids of formula A can be converted to the corresponding ammoniumsalt with an organic salt of formula 5, where X is anion counter ionselected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared accordingto Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to thecorresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) was as follows. A solution of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g). Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the followingscheme 3:

Synthesis of 515

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1L), was added a solution of 514 (10 g,0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere.After complete addition, reaction mixture was warmed to room temperatureand then heated to reflux for 4 h. Progress of the reaction wasmonitored by TLC. After completion of reaction (by TLC) the mixture wascooled to 0 0 C and quenched with careful addition of saturated Na2SO4solution. Reaction mixture was stirred for 4 h at room temperature andfiltered off. Residue was washed well with THF. The filtrate andwashings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCland stirred for 20 minutes at room temperature. The volatilities werestripped off under vacuum to furnish the hydrochloride salt of 515 as awhite solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H),5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO3solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m,2H). LC-MS [M+H] −232.3 (96.94%).

Synthesis of 517A and 517B

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na2SO3 and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50mL). Organic phase was dried over an.Na2SO4 and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield: −6 g crude 517A—Peak-1 (white solid), 5.13 g (96%).1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m,1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m,4H). LC-MS—[M+H]-266.3, [M+NH4+]−283.5 present, HPLC-97.86%.Stereochemistry confirmed by X-ray.

Synthesis of 518

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil.1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H),5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519

A solution of compound 518 (1 eq) in hexane (15 mL) was added in adrop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 400° C. over 0.5 hthen cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na2SO4 then filtered through celite and reducedto an oil. Column chromatography provided the pure 519 (1.3 g, 68%)which was obtained as a colorless oil. 13C NMR 6=130.2, 130.1 (×2),127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7,29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1;Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc.654.6, Found 654.6.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totaldsRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated dsRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” dsRNA content (as measured by thesignal in the absence of surfactant) from the total dsRNA content.Percent entrapped dsRNA is typically >85%. For LNP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancersurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention can be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion canbe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention can be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

An RNAi agent of the invention may be incorporated into a particle,e.g., a microparticle. Microparticles can be produced by spray-drying,but may also be produced by other methods including lyophilization,evaporation, fluid bed drying, vacuum drying, or a combination of thesetechniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of iRNAs through themucosa is enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (seee.g., Malmsten, M. Surfactants and polymers in drug delivery, InformaHealth Care, New York, N.Y., 2002; Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of iRNAsthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Suitable chelating agents include but are not limited todisodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(see e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of iRNAs through the alimentary mucosa (see e.g.,Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers includes, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a)D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or moreagents which function by a non-RNAi mechanism and which are useful intreating a bleeding disorder. Examples of such agents include, but arenot limited to an anti-inflammatory agent, anti-steatosis agent,anti-viral, and/or anti-fibrosis agent. In addition, other substancescommonly used to protect the liver, such as silymarin, can also be usedin conjunction with the iRNAs described herein. Other agents useful fortreating liver diseases include telbivudine, entecavir, and proteaseinhibitors such as telaprevir and other disclosed, for example, in Tunget al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116,and 2003/0144217; and in Hale et al., U.S. Application Publication No.2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED50 with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby Serpina1 expression. In any event, the administering physician canadjust the amount and timing of iRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

IV. Methods for Inhibiting Serpina1 Expression

The present invention provides methods of inhibiting expression of aSerpina1 in a cell. The methods include contacting a cell with an RNAiagent, e.g., a double stranded RNAi agent, in an amount effective toinhibit expression of the Serpina1 in the cell, thereby inhibitingexpression of the Serpina1 in the cell.

Contacting of a cell with a double stranded RNAi agent may be done invitro or in vivo. Contacting a cell in vivo with the RNAi agent includescontacting a cell or group of cells within a subject, e.g., a humansubject, with the RNAi agent. Combinations of in vitro and in vivomethods of contacting are also possible. Contacting may be direct orindirect, as discussed above. Furthermore, contacting a cell may beaccomplished via a targeting ligand, including any ligand describedherein or known in the art. In preferred embodiments, the targetingligand is a carbohydrate moiety, e.g., a GalNAc₃ ligand, or any otherligand that directs the RNAi agent to a site of interest, e.g., theliver of a subject.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating” and other similar terms, andincludes any level of inhibition.

The phrase “inhibiting expression of a Serpina1” is intended to refer toinhibition of expression of any Serpina1 gene (such as, e.g., a mouseSerpina1 gene, a rat Serpina1 gene, a monkey Serpina1 gene, or a humanSerpina1 gene) as well as variants or mutants of a Serpina1 gene. Thus,the Serpina1 gene may be a wild-type Serpina1 gene, a mutant Serpina1gene, or a transgenic Serpina1 gene in the context of a geneticallymanipulated cell, group of cells, or organism.

“Inhibiting expression of a Serpina1 gene” includes any level ofinhibition of a Serpina1 gene, e.g., at least partial suppression of theexpression of a Serpina1 gene. The expression of the Serpina1 gene maybe assessed based on the level, or the change in the level, of anyvariable associated with Serpina1 gene expression, e.g., Serpina1 mRNAlevel, Serpina1 protein level, or lipid levels. This level may beassessed in an individual cell or in a group of cells, including, forexample, a sample derived from a subject.

Inhibition may be assessed by a decrease in an absolute or relativelevel of one or more variables that are associated with Serpina1expression compared with a control level. The control level may be anytype of control level that is utilized in the art, e.g., a pre-dosebaseline level, or a level determined from a similar subject, cell, orsample that is untreated or treated with a control (such as, e.g.,buffer only control or inactive agent control).

In some embodiments of the methods of the invention, expression of aSerpina1 gene is inhibited by at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%. at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99%.

Inhibition of the expression of a Serpina1 gene may be manifested by areduction of the amount of mRNA expressed by a first cell or group ofcells (such cells may be present, for example, in a sample derived froma subject) in which a Serpina1 gene is transcribed and which has or havebeen treated (e.g., by contacting the cell or cells with an RNAi agentof the invention, or by administering an RNAi agent of the invention toa subject in which the cells are or were present) such that theexpression of a Serpina1 gene is inhibited, as compared to a second cellor group of cells substantially identical to the first cell or group ofcells but which has not or have not been so treated (control cell(s)).In preferred embodiments, the inhibition is assessed by expressing thelevel of mRNA in treated cells as a percentage of the level of mRNA incontrol cells, using the following formula:

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, inhibition of the expression of a Serpina1 gene may beassessed in terms of a reduction of a parameter that is functionallylinked to Serpina1 gene expression, e.g., Serpina1 protein expression,such as ALT, alkaline phosphatase, bilirubin, prothrombin and albumin.Serpina1 gene silencing may be determined in any cell expressingSerpina1, either constitutively or by genomic engineering, and by anyassay known in the art. The liver is the major site of Serpina1expression. Other significant sites of expression include the lung andintestines.

Inhibition of the expression of a Serpina1 protein may be manifested bya reduction in the level of the Serpina1 protein that is expressed by acell or group of cells (e.g., the level of protein expressed in a samplederived from a subject). As explained above for the assessment of mRNAsuppression, the inhibition of protein expression levels in a treatedcell or group of cells may similarly be expressed as a percentage of thelevel of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess theinhibition of the expression of a Serpina1 gene includes a cell or groupof cells that has not yet been contacted with an RNAi agent of theinvention. For example, the control cell or group of cells may bederived from an individual subject (e.g., a human or animal subject)prior to treatment of the subject with an RNAi agent.

The level of Serpina1 mRNA that is expressed by a cell or group of cellsmay be determined using any method known in the art for assessing mRNAexpression. In one embodiment, the level of expression of Serpina1 in asample is determined by detecting a transcribed polynucleotide, orportion thereof, e.g., mRNA of the Serpina1 gene. RNA may be extractedfrom cells using RNA extraction techniques including, for example, usingacid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis),RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix,Switzerland). Typical assay formats utilizing ribonucleic acidhybridization include nuclear run-on assays, RT-PCR, RNase protectionassays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, insitu hybridization, and microarray analysis.

In one embodiment, the level of expression of Serpina1 is determinedusing a nucleic acid probe. The term “probe”, as used herein, refers toany molecule that is capable of selectively binding to a specificSerpina1. Probes can be synthesized by one of skill in the art, orderived from appropriate biological preparations. Probes may bespecifically designed to be labeled. Examples of molecules that can beutilized as probes include, but are not limited to, RNA, DNA, proteins,antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or Northern analyses,polymerase chain reaction (PCR) analyses and probe arrays. One methodfor the determination of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize to Serpina1mRNA. In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in an Affymetrix gene chip array. A skilledartisan can readily adapt known mRNA detection methods for use indetermining the level of Serpina1 mRNA.

An alternative method for determining the level of expression ofSerpina1 in a sample involves the process of nucleic acid amplificationand/or reverse transcriptase (to prepare cDNA) of for example mRNA inthe sample, e.g., by RT-PCR (the experimental embodiment set forth inMullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany(1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardiet al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers. Inparticular aspects of the invention, the level of expression of Serpina1is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™System).

The expression levels of Serpina1 mRNA may be monitored using a membraneblot (such as used in hybridization analysis such as Northern, Southern,dot, and the like), or microwells, sample tubes, gels, beads or fibers(or any solid support comprising bound nucleic acids). See U.S. Pat.Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which areincorporated herein by reference. The determination of Serpina1expression level may also comprise using nucleic acid probes insolution.

In preferred embodiments, the level of mRNA expression is assessed usingbranched DNA (bDNA) assays or real time PCR (qPCR). The use of thesemethods is described and exemplified in the Examples presented herein.

The level of Serpina1 protein expression may be determined using anymethod known in the art for the measurement of protein levels. Suchmethods include, for example, electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, fluid or gelprecipitin reactions, absorption spectroscopy, a colorimetric assays,spectrophotometric assays, flow cytometry, immunodiffusion (single ordouble), immunoelectrophoresis, Western blotting, radioimmunoassay(RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescentassays, electrochemiluminescence assays, and the like.

The term “sample” as used herein refers to a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, lymph, urine,cerebrospinal fluid, saliva, ocular fluids, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In preferred embodiments, a “sample derived from asubject” refers to blood or plasma drawn from the subject. In furtherembodiments, a “sample derived from a subject” refers to liver tissuederived from the subject.

In some embodiments of the methods of the invention, the RNAi agent isadministered to a subject such that the RNAi agent is delivered to aspecific site within the subject. The inhibition of expression ofSerpina1 may be assessed using measurements of the level or change inthe level of Serpina1 mRNA or Serpina1 protein in a sample derived fromfluid or tissue from the specific site within the subject. In preferredembodiments, the site is the liver. The site may also be a subsection orsubgroup of cells from any one of the aforementioned sites. The site mayalso include cells that express a particular type of receptor.

V. Methods for Treating or Preventing a Serpina1 Associated Disease

The present invention also provides methods for treating or preventingdiseases and conditions that can be modulated by down regulatingSerpina1 gene expression. For example, the compositions described hereincan be used to treat Serpina1 associated diseases, such as liverdiseases, e.g., chronic liver disease, liver inflammation, cirrhosis,liver fibrosis, and/or hepatocellular carcinoma, and other pathologicalconditions that may be associated with these disorders, such as lunginflammation, emphysema, and COPD.

The present invention also provides methods for inhibiting thedevelopment of hepatocellular carcinoma in a subject, e.g., a subjecthaving a Serpina1 deficiency variant. The methods include administeringa therapeutically effective amount of a composition of the invention tothe subject, thereby inhibiting the development of hepatocellularcarcinoma in the subject.

Methods and uses of the compositions of the invention for reducing theaccumulation of misfolded Serpina1 in the liver of a subject, e.g., asubject having a Serpina1 deficiency variant, are also provided by thepresent invention. The methods include administering a therapeuticallyeffective amount of a composition of the invention to the subject,thereby reducing the accumulation of misfolded Serpina1 in the liver ofthe subject.

As used herein, a “subject” includes a human or non-human animal,preferably a vertebrate, and more preferably a mammal. A subject mayinclude a transgenic organism. Most preferably, the subject is a human,such as a human suffering from or predisposed to developing aSerpina1-associated disease. In one embodiment, the subject suffering orpredisposed to developing a Serpina1-associated disease has one or moreSerpina1 deficient alleles, e.g., a PIZ, PIS, or PIM(Malton) allele.

In further embodiments of the invention, an iRNA agent of the inventionis administered in combination with an additional therapeutic agent. TheiRNA agent and an additional therapeutic agent can be administered incombination in the same composition, e.g., parenterally, or theadditional therapeutic agent can be administered as part of a separatecomposition or by another method described herein.

Examples of additional therapeutic agents suitable for use in themethods of the invention include those agents known to treat liverdisorders, such as liver cirhosis. For example, an iRNA agent featuredin the invention can be administered with, e.g., ursodeoxycholic acid(UDCA), immunosuppressive agents, methotrexate, corticosteroids,cyclosporine, colchicine, antipruritic treatments, such asantihistamines, cholestyramine, colestipol, rifampin, dronabinol(Marinol), and plasmaphesesis, prophylactic antibiotics, ultravioletlight, zinc supplements, and hepatitis A, influenza and pneumococcivaccination.

In some embodiments of the methods of the invention, Serpina1 expressionis decreased for an extended duration, e.g., at least one week, twoweeks, three weeks, or four weeks or longer. For example, in certaininstances, expression of the Serpina1 gene is suppressed by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% byadministration of an iRNA agent described herein. In some embodiments,the Serpina1 gene is suppressed by at least about 60%, 70%, or 80% byadministration of the iRNA agent. In some embodiments, the Serpina1 geneis suppressed by at least about 85%, 90%, or 95% by administration ofthe iRNA agent.

The iRNA agents of the invention may be administered to a subject usingany mode of administration known in the art, including, but not limitedto subcutaneous, intravenous, intramuscular, intraocular,intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic,cerebrospinal, and any combinations thereof. In preferred embodiments,the iRNA agents are administered subcutaneously.

In some embodiments, the administration is via a depot injection. Adepot injection may release the iRNA agents in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof Serpina1, or a therapeutic or prophylactic effect. A depot injectionmay also provide more consistent serum concentrations. Depot injectionsmay include subcutaneous injections or intramuscular injections. Inpreferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the RNAi agent to the liver.

Other modes of administration include epidural, intracerebral,intracerebroventricular, nasal administration, intraarterial,intracardiac, intraosseous infusion, intrathecal, and intravitreal, andpulmonary. The mode of administration may be chosen based upon whetherlocal or systemic treatment is desired and based upon the area to betreated. The route and site of administration may be chosen to enhancetargeting.

The methods of the invention include administering an iRNA agent at adose sufficient to suppress/decrease levels of Serpina1 mRNA for atleast 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days; andoptionally, administering a second single dose of the iRNA agent,wherein the second single dose is administered at least 5, morepreferably 7, 10, 14, 21, 25, 30 or 40 days after the first single doseis administered, thereby inhibiting the expression of the Serpina1 genein a subject.

In one embodiment, doses of an iRNA agent of the invention areadministered not more than once every four weeks, not more than onceevery three weeks, not more than once every two weeks, or not more thanonce every week. In another embodiment, the administrations can bemaintained for one, two, three, or six months, or one year or longer.

In general, the iRNA agent does not activate the immune system, e.g., itdoes not increase cytokine levels, such as TNF-alpha or IFN-alphalevels. For example, when measured by an assay, such as an in vitro PBMCassay, such as described herein, the increase in levels of TNF-alpha orIFN-alpha, is less than 30%, 20%, or 10% of control cells treated with acontrol iRNA agent, such as an iRNA agent that does not target Serpina1.

For example, a subject can be administered a therapeutic amount of aniRNA agent, such as 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5mg/kg dsRNA. The iRNA agent can be administered by intravenous infusionover a period of time, such as over a 5 minute, 10 minute, 15 minute, 20minute, or 25 minute period. The administration is repeated, forexample, on a regular basis, such as biweekly (i.e., every two weeks)for one month, two months, three months, four months or longer.

After an initial treatment regimen, the treatments can be administeredon a less frequent basis. For example, after administration biweekly forthree months, administration can be repeated once per month, for sixmonths or a year or longer. Administration of the iRNA agent can reduceSerpina1 levels, e.g., in a cell, tissue, blood, urine, organ (e.g., theliver), or other compartment of the patient by at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80% or at least 90% or more.

Before administration of a full dose of the iRNA agent, patients can beadministered a smaller dose, and monitored for adverse effects, such asan allergic reaction, or for elevated lipid levels or blood pressure. Inanother example, the patient can be monitored for unwantedimmunostimulatory effects, such as increased cytokine (e.g., TNF-alphaor INF-alpha) levels. An exemplary smaller dose is one that results inan incidence of infusion reaction of less than or equal to 5%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Forexample, efficacy of treatment of liver fibrosis or amelioration ofliver fibrosis can be assessed, for example by periodic monitoring ofliver fibrosis markers: a-2-macroglobulin(a-MA), transferrin,apolipoproteinAl, hyaluronic acid (HA), laminin, N-terminal procollagenIII(PIIINP), 7S collagen IV (7S-IV), total bilirubin, indirectbilirubin, alanine aminotransferase (ALT), aspartate aminotransferase(AST), AST/ALT, g-glutamyl transpeptidase (GGT), alkaline phosphatase(ALP), albumin, albumin/globulin, blood urea nitrogen (BUN), creatinine(Cr), triglyceride, cholersterol, high density lipoprotein and lowdensity lipoprotein and liver puncture biopsy. Liver fibrosis markerscan be measured and/or liver puncture biopsy can be performed beforetreatment (initial readings) and subsequently (later readings) duringthe treatment regimen.

Comparisons of the later readings with the initial readings provide aphysician an indication of whether the treatment is effective. It iswell within the ability of one skilled in the art to monitor efficacy oftreatment or prevention by measuring any one of such parameters, or anycombination of parameters. In connection with the administration of aniRNA agent targeting Serpina1 or pharmaceutical composition thereof,“effective against” a Serpina1 associate disease, such as a liverdisease, e.g., a hepatic fibrosis condition, indicates thatadministration of an iRNA agent of the invention in a clinicallyappropriate manner results in a beneficial effect for at least astatistically significant fraction of patients, such as an improvementof symptoms, a cure, a reduction in disease load, reduction in tumormass or cell numbers, extension of life, improvement in quality of life,or other effect generally recognized as positive by medical doctorsfamiliar with treating liver diseases.

In the methods of the invention, an iRNA agent as described herein canbe used to treat individuals having the signs, symptoms and/or markersof, or being diagnosed with, or being a risk of having an Serpina1associate disease, such as a liver disease, e.g., liver inflammation,cirrhosis, liver fibrosis, and/or hepatoceullar carcinoma. One of skillin the art can easily monitor the signs, symptoms, and/or makers of suchdisorders in subjects receiving treatment with an iRNA agent asdescribed herein and assay for a reduction in these signs, symptomsand/or makers of at least 10% and preferably to a clinical levelrepresenting a low risk of liver disease.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease (such as a liverfunction described supra), and preferably at least 20%, 30%, 40%, 50% ormore can be indicative of effective treatment. Efficacy for a given iRNAagent of the invention or formulation of that iRNA agent can also bejudged using an experimental animal model for the given disease as knownin the art. When using an experimental animal model, efficacy oftreatment is evidenced when a statistically significant reduction in amarker or symptom is observed.

A treatment or preventive effect is also evident when one or moresymptoms are reduced or alleviated. For example, a treatment orpreventive is effective when one or more of weakness, fatigue, weightloss, nausea, vomiting, abdominal swelling, extremity swelling,excessive itching, and jaundice of the eyes and/or skin is reduced oralleviated.

For certain indications, the efficacy can be measured by an increase inserum levels of Serpina1 protein. As an example, an increase of serumlevels of properly folded Serpina1 of at least 10%, at least 20%, atleast 50%, at least 100%, at least 200% more can be indicative ofeffective treatment.

Alternatively, the efficacy can be measured by a reduction in theseverity of disease as determined by one skilled in the art of diagnosisbased on a clinically accepted disease severity grading scale, as butone example the Child-Pugh score (sometimes the Child-Turcotte-Pughscore). In this example, prognosis of chronic liver disease, mainlycirrhosis, is measured by an aggregate score of five clinical measures,billirubin, serum albumin, INR, ascites, and hepatic encephalopathy.Each marker is assigned a value from 1-3, and the total value is used toprovide a score categorized as A (5-6 points), B (7-9 points), or C(10-15 points), which can be correlated with one and two year survivalrates. Methods for determination and analysis of Child-Pugh scores arewell known in the art (Farnsworth et al, Am J Surgery 2004 188:580-583;Child and Turcotte. Surgery and portal hypertension. In: The liver andportal hypertension. Edited by CG Child. Philadelphia: Saunders1964:50-64; Pugh et al, Br J Surg 1973; 60:648-52). Efficacy can bemeasured in this example by the movement of a patient from e.g., a “B”to an “A.” Any positive change resulting in e.g., lessening of severityof disease measured using the appropriate scale, represents adequatetreatment using an iRNA or iRNA formulation as described herein.

In one embodiment, the RNAi agent is administered at a dose of betweenabout 0.25 mg/kg to about 50 mg/kg, e.g., between about 0.25 mg/kg toabout 0.5 mg/kg, between about 0.25 mg/kg to about 1 mg/kg, betweenabout 0.25 mg/kg to about 5 mg/kg, between about 0.25 mg/kg to about 10mg/kg, between about 1 mg/kg to about 10 mg/kg, between about 5 mg/kg toabout 15 mg/kg, between about 10 mg/kg to about 20 mg/kg, between about15 mg/kg to about 25 mg/kg, between about 20 mg/kg to about 30 mg/kg,between about 25 mg/kg to about 35 mg/kg, or between about 40 mg/kg toabout 50 mg/kg.

In some embodiments, the RNAi agent is administered at a dose of about0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg,about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17mg/kg, about 18 mg/kg, about 19 mg/kg, about 20 mg/kg, about 21 mg/kg,about 22 mg/kg, about 23 mg/kg, about 24 mg/kg, about 25 mg/kg, about 26mg/kg, about 27 mg/kg, about 28 mg/kg, about 29 mg/kg, 30 mg/kg, about31 mg/kg, about 32 mg/kg, about 33 mg/kg, about 34 mg/kg, about 35mg/kg, about 36 mg/kg, about 37 mg/kg, about 38 mg/kg, about 39 mg/kg,about 40 mg/kg, about 41 mg/kg, about 42 mg/kg, about 43 mg/kg, about 44mg/kg, about 45 mg/kg, about 46 mg/kg, about 47 mg/kg, about 48 mg/kg,about 49 mg/kg or about 50 mg/kg.

In certain embodiments of the invention, for example, when adouble-stranded RNAi agent includes modifications (e.g., one or moremotifs of three identical modifications on three consecutivenucleotides, including one such motif at or near the cleavage site ofthe agent), six phosphorothioate linkages, and a ligand, such an agentis administered at a dose of about 0.01 to about 0.5 mg/kg, about 0.01to about 0.4 mg/kg, about 0.01 to about 0.3 mg/kg, about 0.01 to about0.2 mg/kg, about 0.01 to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09mg/kg, about 0.01 mg/kg to about 0.08 mg/kg, about 0.01 mg/kg to about0.07 mg/kg, about 0.01 mg/kg to about 0.06 mg/kg, about 0.01 mg/kg toabout 0.05 mg/kg, about 0.02 to about 0.5 mg/kg, about 0.02 to about 0.4mg/kg, about 0.02 to about 0.3 mg/kg, about 0.02 to about 0.2 mg/kg,about 0.02 to about 0.1 mg/kg, about 0.02 mg/kg to about 0.09 mg/kg,about 0.02 mg/kg to about 0.08 mg/kg, about 0.02 mg/kg to about 0.07mg/kg, about 0.02 mg/kg to about 0.06 mg/kg, about 0.02 mg/kg to about0.05 mg/kg, about 0.03 to about 0.5 mg/kg, about 0.03 to about 0.4mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg,about 0.03 to about 0.1 mg/kg, about 0.03 mg/kg to about 0.09 mg/kg,about 0.03 mg/kg to about 0.08 mg/kg, about 0.03 mg/kg to about 0.07mg/kg, about 0.03 mg/kg to about 0.06 mg/kg, about 0.03 mg/kg to about0.05 mg/kg, about 0.04 to about 0.5 mg/kg, about 0.04 to about 0.4mg/kg, about 0.04 to about 0.3 mg/kg, about 0.04 to about 0.2 mg/kg,about 0.04 to about 0.1 mg/kg, about 0.04 mg/kg to about 0.09 mg/kg,about 0.04 mg/kg to about 0.08 mg/kg, about 0.04 mg/kg to about 0.07mg/kg, about 0.04 mg/kg to about 0.06 mg/kg, about 0.05 to about 0.5mg/kg, about 0.05 to about 0.4 mg/kg, about 0.05 to about 0.3 mg/kg,about 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.05mg/kg to about 0.09 mg/kg, about 0.05 mg/kg to about 0.08 mg/kg, orabout 0.05 mg/kg to about 0.07 mg/kg. Values and ranges intermediate tothe foregoing recited values are also intended to be part of thisinvention, e.g., the RNAi agent may be administered to the subject at adose of about 0.015 mg/kg to about 0.45 mg/mg.

For example, the RNAi agent, e.g., RNAi agent in a pharmaceuticalcomposition, may be administered at a dose of about 0.01 mg/kg, 0.0125mg/kg, 0.015 mg/kg, 0.0175 mg/kg, 0.02 mg/kg, 0.0225 mg/kg, 0.025 mg/kg,0.0275 mg/kg, 0.03 mg/kg, 0.0325 mg/kg, 0.035 mg/kg, 0.0375 mg/kg, 0.04mg/kg, 0.0425 mg/kg, 0.045 mg/kg, 0.0475 mg/kg, 0.05 mg/kg, 0.0525mg/kg, 0.055 mg/kg, 0.0575 mg/kg, 0.06 mg/kg, 0.0625 mg/kg, 0.065 mg/kg,0.0675 mg/kg, 0.07 mg/kg, 0.0725 mg/kg, 0.075 mg/kg, 0.0775 mg/kg, 0.08mg/kg, 0.0825 mg/kg, 0.085 mg/kg, 0.0875 mg/kg, 0.09 mg/kg, 0.0925mg/kg, 0.095 mg/kg, 0.0975 mg/kg, 0.1 mg/kg, 0.125 mg/kg, 0.15 mg/kg,0.175 mg/kg, 0.2 mg/kg, 0.225 mg/kg, 0.25 mg/kg, 0.275 mg/kg, 0.3 mg/kg,0.325 mg/kg, 0.35 mg/kg, 0.375 mg/kg, 0.4 mg/kg, 0.425 mg/kg, 0.45mg/kg, 0.475 mg/kg, or about 0.5 mg/kg. Values intermediate to theforegoing recited values are also intended to be part of this invention.

The dose of an RNAi agent that is administered to a subject may betailored to balance the risks and benefits of a particular dose, forexample, to achieve a desired level of Serpina1 gene suppression (asassessed, e.g., based on Serpina1 mRNA suppression, Serpina1 proteinexpression) or a desired therapeutic or prophylactic effect, while atthe same time avoiding undesirable side effects.

In some embodiments, the RNAi agent is administered in two or moredoses. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable. In some embodiments, the number or amountof subsequent doses is dependent on the achievement of a desired effect,e.g., the suppression of a Serpina1 gene, or the achievement of atherapeutic or prophylactic effect, e.g., reducing a symptom of a liverdisease. In some embodiments, the RNAi agent is administered accordingto a schedule. For example, the RNAi agent may be administered once perweek, twice per week, three times per week, four times per week, or fivetimes per week. In some embodiments, the schedule involves regularlyspaced administrations, e.g., hourly, every four hours, every six hours,every eight hours, every twelve hours, daily, every 2 days, every 3days, every 4 days, every 5 days, weekly, biweekly, or monthly. In otherembodiments, the schedule involves closely spaced administrationsfollowed by a longer period of time during which the agent is notadministered. For example, the schedule may involve an initial set ofdoses that are administered in a relatively short period of time (e.g.,about every 6 hours, about every 12 hours, about every 24 hours, aboutevery 48 hours, or about every 72 hours) followed by a longer timeperiod (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks,about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks) duringwhich the RNAi agent is not administered. In one embodiment, the RNAiagent is initially administered hourly and is later administered at alonger interval (e.g., daily, weekly, biweekly, or monthly). In anotherembodiment, the RNAi agent is initially administered daily and is lateradministered at a longer interval (e.g., weekly, biweekly, or monthly).In certain embodiments, the longer interval increases over time or isdetermined based on the achievement of a desired effect. In a specificembodiment, the RNAi agent is administered once daily during a firstweek, followed by weekly dosing starting on the eighth day ofadministration. In another specific embodiment, the RNAi agent isadministered every other day during a first week followed by weeklydosing starting on the eighth day of administration.

In some embodiments, the RNAi agent is administered in a dosing regimenthat includes a “loading phase” of closely spaced administrations thatmay be followed by a “maintenance phase”, in which the RNAi agent isadministered at longer spaced intervals. In one embodiment, the loadingphase comprises five daily administrations of the RNAi agent during thefirst week. In another embodiment, the maintenance phase comprises oneor two weekly administrations of the RNAi agent. In a furtherembodiment, the maintenance phase lasts for 5 weeks. In one embodiment,the loading phase comprises administration of a dose of 2 mg/kg, 1 mg/kgor 0.5 mg/kg five times a week. In another embodiment, the maintenancephase comprises administration of a dose of 2 mg/kg, 1 mg/kg or 0.5mg/kg once or twice weekly.

Any of these schedules may optionally be repeated for one or moreiterations. The number of iterations may depend on the achievement of adesired effect, e.g., the suppression of a Serpina1 gene, and/or theachievement of a therapeutic or prophylactic effect, e.g., reducing asymptom of a Serpina1 associated disease, e.g., a liver disease.

In another aspect, the invention features, a method of instructing anend user, e.g., a caregiver or a subject, on how to administer an iRNAagent described herein. The method includes, optionally, providing theend user with one or more doses of the iRNA agent, and instructing theend user to administer the iRNA agent on a regimen described herein,thereby instructing the end user.

Genetic predisposition plays a role in the development of target geneassociated diseases, e.g., liver disease. Therefore, a patient in needof a siRNA can be identified by taking a family history, or, forexample, screening for one or more genetic markers or variants.Accordingly, in one aspect, the invention provides a method of treatinga patient by selecting a patient on the basis that the patient has oneor more of a Serpina1 deficiency or a Serpina1 deficiency gene variant,e.g., a PIZ, PIS, or PIM(Malton) allele. The method includesadministering to the patient an iRNA agent in a therapeuticallyeffective amount.

A healthcare provider, such as a doctor, nurse, or family member, cantake a family history before prescribing or administering an iRNA agentof the invention. In addition, a test may be performed to determine ageneotype or phenotype. For example, a DNA test may be performed on asample from the patient, e.g., a blood sample, to identify the Serpina1genotype and/or phenotype before a Serpina1 dsRNA is administered to thepatient.

VI. Kits

The present invention also provides kits for using any of the iRNAagents and/or performing any of the methods of the invention. Such kitsinclude one or more RNAi agent(s) and instructions for use, e.g.,instructions for inhibiting expression of a Serpina1 in a cell bycontacting the cell with the RNAi agent(s) in an amount effective toinhibit expression of the Serpina1. The kits may optionally furthercomprise means for contacting the cell with the RNAi agent (e.g., aninjection device), or means for measuring the inhibition of Serpina1(e.g., means for measuring the inhibition of Serpina1 mRNA). Such meansfor measuring the inhibition of Serpina1 may comprise a means forobtaining a sample from a subject, such as, e.g., a plasma sample. Thekits of the invention may optionally further comprise means foradministering the RNAi agent(s) to a subject or means for determiningthe therapeutically effective or prophylactically effective amount.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the iRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Materials and Methods

The following materials and methods were used in the Examples.

siRNA Design

The Serpina1 gene has multiple, alternate transcripts. siRNA design wascarried out to identify siRNAs targeting all human and Cynomolgus monkey(Macaca fascicularis; henceforth “cyno”) Serpina1 transcripts annotatedin the NCBI Gene database (http://www.ncbi.nlm.nih.gov/gene/). Thefollowing human transcripts from the NCBI RefSeq collection were used:Human—NM 000295.4, NM_001002235.2, NM_001002236.2, NM_001127700.1,NM_001127701.1, NM_001127702.1, NM_001127703.1, NM_001127704.1,NM_001127705.1, NM_001127706.1, NM_001127707.1. To identify a cynotranscript, the rhesus monkey (Macaca mulatta) transcript,XM_001099255.2, was aligned to the M. fascicularis genome using theSpidey alignment tool (www.ncbi.nlm.nih.gov/spidey). The overall percentidentity of rhesus and cyno transcripts was 99.6%. The cyno transcriptwas hand-assembled to preserve consensus splice sites and full-lengthcoding and untranslated regions. The resulting transcript was 2064nucleotides long.

All siRNA duplexes were designed that shared 100% identity with alllisted human and cyno transcripts.

Five hungered eighty-five candidate siRNAs were used in a comprehensivesearch against the human transcriptome (defined as the set of NM_andXM_records within the human NCBI Refseq set). A total of 48 sense (21mers) and 48 antisense (23 mers) derived siRNA oligos were synthesizedand formed into duplexes. A detailed list of Sepina1 sense and antisensestrand sequences is shown in Tables 1 and 2.

siRNA Synthesis

I. General Small and Medium Scale RNA Synthesis Procedure

RNA oligonucleotides were synthesized at scales between 0.2-500 μmolusing commercially available5′-O-(4,4′-dimethoxytrityl)-2′-O-t-butyldimethylsilyl-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramiditemonomers of uridine, 4-N-acetylcytidine, 6-N-benzoyladenosine and2-N-isobutyrylguanosine and the corresponding 2′-O-methyl and 2′-fluorophosphoramidites according to standard solid phase oligonucleotidesynthesis protocols. The amidite solutions were prepared at 0.1-0.15 Mconcentration and 5-ethylthio-1H-tetrazole (0.25-0.6 M in acetonitrile)was used as the activator. Phosphorothioate backbone modifications wereintroduced during synthesis using 0.2 M phenylacetyl disulfide (PADS) inlutidine:acetonitrile (1:1) (v;v) or 0.1 M 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) in pyridine for the oxidationstep. After completion of synthesis, the sequences were cleaved from thesolid support and deprotected using methylamine followed bytriethylamine.3HF to remove any 2′-O-t-butyldimethylsilyl protectinggroups present.

For synthesis scales between 5-500 μmol and fully 2′ modified sequences(2′-fluoro and/or 2′-O-methyl or combinations thereof) theoligonucleotides where deprotected using 3:1 (v/v) ethanol andconcentrated (28-32%) aqueous ammonia either at 35° C. 16 h or 55° C.for 5.5 h. Prior to ammonia deprotection the oligonucleotides wheretreated with 0.5 M piperidine in acetonitrile for 20 min on the solidsupport. The crude oligonucleotides were analyzed by LC-MS andanion-exchange HPLC (IEX-HPLC). Purification of the oligonucleotides wascarried out by IEX HPLC using: 20 mM phosphate, 10%-15% ACN, pH=8.5(buffer A) and 20 mM phosphate, 10%-15% ACN, 1 M NaBr, pH=8.5 (bufferB). Fractions were analyzed for purity by analytical HPLC. Theproduct-containing fractions with suitable purity were pooled andconcentrated on a rotary evaporator prior to desalting. The samples weredesalted by size exclusion chromatography and lyophilized to dryness.Equal molar amounts of sense and antisense strands were annealed in1×PBS buffer to prepare the corresponding siRNA duplexes.

For small scales (0.2-1 μmol), synthesis was performed on a MerMade 192synthesizer in a 96 well format. In case of fully 2′-modified sequences(2′-fluoro and/or 2′-O-methyl or combinations thereof) theoligonucleotides where deprotected using methylamine at room temperaturefor 30-60 min followed by incubation at 60° C. for 30 min or using 3:1(v/v) ethanol and concentrated (28-32%) aqueous ammonia at roomtemperature for 30-60 min followed by incubation at 40° C. for 1.5hours. The crude oligonucleotides were then precipitated in a solutionof acetonitrile:acetone (9:1) and isolated by centrifugation anddecanting the supernatant. The crude oligonucleotide pellet wasre-suspended in 20 mM NaOAc buffer and analyzed by LC-MS and anionexchange HPLC. The crude oligonucleotide sequences were desalted in 96deep well plates on a 5 mL HiTrap Sephadex G25 column (GE Healthcare).In each well about 1.5 mL samples corresponding to an individualsequence was collected. These purified desalted oligonucleotides wereanalyzed by LC-MS and anion exchange chromatography. Duplexes wereprepared by annealing equimolar amounts of sense and antisense sequenceson a Tecan robot. Concentration of duplexes was adjusted to 10 μM in1×PBS buffer.

II. Synthesis of GalNAc-Conjugated Oligonucleotides for In Vivo Analysis

Oligonucleotides conjugated with GalNAc ligand at their 3′-terminus weresynthesized at scales between 0.2-500 μmol using a solid supportpre-loaded with a Y-shaped linker bearing a 4,4′-dimethoxytrityl(DMT)-protected primary hydroxy group for oligonucleotide synthesis anda GalNAc ligand attached through a tether.

For synthesis of GalNAc conjugates in the scales between 5-500 μmol, theabove synthesis protocol for RNA was followed with the followingadaptions: For polystyrene-based synthesis supports 5% dichloroaceticacid in toluene was used for DMT-cleavage during synthesis. Cleavagefrom the support and deprotection was performed as described above.Phosphorothioate-rich sequences (usually >5 phorphorothioates) weresynthesized without removing the final 5′-DMT group (“DMT-on”) and,after cleavage and deprotection as described above, purified by reversephase HPLC using 50 mM ammonium acetate in water (buffer A) and 50 mMammoniumacetate in 80% acetonitirile (buffer B). Fractions were analyzedfor purity by analytical HPLC and/or LC-MS. The product-containingfractions with suitable purity were pooled and concentrated on a rotaryevaporator. The DMT-group was removed using 20%-25% acetic acid in wateruntil completion. The samples were desalted by size exclusionchromatography and lyophilized to dryness. Equal molar amounts of senseand antisense strands were annealed in 1×PBS buffer to prepare thecorresponding siRNA duplexes.

For small scale synthesis of GalNAc conjugates (0.2-1 μmol), includingsequences with multiple phosphorothioate linkages, the protocolsdescribed above for synthesis of RNA or fully 2′-F/2′-OMe-containingsequences on MerMade platform were applied. Synthesis was performed onpre-packed columns containing GalNAc-functionalized controlled poreglass support.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H₂O perreaction was added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Cell Culture and Transfections

Hep3B, HepG2 or HeLa cells (ATCC, Manassas, Va.) were grown to nearconfluence at 37° C. in an atmosphere of 5% CO₂ in recommended media(ATCC) supplemented with 10% FBS and glutamine (ATCC) before beingreleased from the plate by trypsinization. For duplexes screened in96-well format, transfection was carried out by adding 44.75 μl ofOpti-MEM plus 0.25 μl of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) to 5 μl of each siRNA duplex to anindividual well in a 96-well plate. The mixture was then incubated atroom temperature for 15 minutes. Fifty μl of complete growth mediawithout antibiotic containing ˜2×10⁴ cells were then added to the siRNAmixture. For duplexes screened in 384-well format, 5 μl of Opti-MEM plus0.1 μl of Lipofectamine RNAiMax (Invitrogen, Carlsbad Calif. cat#13778-150) was mixed with 5 μl of each siRNA duplex per an individualwell. The mixture was then incubated at room temperature for 15 minutesfollowed by addition of 40 μl of complete growth media withoutantibiotic containing ˜8×10³ cells. Cells were incubated for 24 hoursprior to RNA purification. Single dose experiments were performed at 10nM and 0.1 nM final duplex concentration and dose response experimentswere done at 10, 1.67, 0.27, 0.046, 0.0077, 0.0013, 0.00021, 0.00004 nMfinal duplex concentration.

Free Uptake Transfection

Five μl of each GalNac conjugated siRNA in PBS was combined with 3×10⁴freshly thawed cryopreserved Cynomolgus monkey hepatocytes (In VitroTechnologies-Celsis, Baltimore, Md.; lot#JQD) resuspended in 95 μl of InVitro Gro CP media (In Vitro Technologies-Celsis, Baltimore, Md.) ineach well of a 96-well plate or 5 ul siRNA and 45 μl media containing1.2×10³ cells for 384 well plate format. The mixture was incubated forabout 24 hours at 37° C. in an atmosphere of 5% CO₂. siRNAs were testedat final concentrations of 500 nM and 10 nM.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12)

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (themixing speed was the same throughout the process). Ten microliters ofmagnetic beads and 80 μl Lysis/Binding Buffer mixture were added to around bottom plate and mixed for 1 minute. Magnetic beads were capturedusing magnetic stand and the supernatant was removed without disturbingthe beads. After removing the supernatant, the lysed cells were added tothe remaining beads and mixed for 5 minutes. After removing thesupernatant, magnetic beads were washed 2 times with 150 μl Wash BufferA and mixed for 1 minute. Beads were captured again and the supernatantremoved. Beads were then washed with 150 μl Wash Buffer B, captured andthe supernatant was removed. Beads were next washed with 150 μl ElutionBuffer, captured and the supernatant removed. Beads were allowed to dryfor 2 minutes. After drying, 50 μl of Elution Buffer was added and mixedfor 5 minutes at 70° C. Beads were captured on a magnet for 5 minutes.Fifty μl of supernatant was removed and added to another 96-well plate.

For 384-well format, the cells were lysed for one minute by addition of50 μl Lysis/Binding buffer. Two μl of magnetic beads per well was used.The required volume of beads was aliquoted, captured on a magneticstand, and the bead storage solution was removed. The beads were thenresuspended in the required volume of Lysis/Binding buffer (25 μl perwell) and 25 μl of bead suspension was added to the lysed cells. Thelysate-bead mixture was incubated for 10 minutes on VibraTransaltor atsetting #7 (UnionScientific Corp., Randallstown, Md.). Subsequentlybeads were captured using a magnetic stand, the supernatant removed andthe beads are washed once with 90 μl Buffer A, followed by singlewashing steps with 90 μl Buffer B and 100 μl of Elution buffer. Thebeads were soaked in each washing buffer for ˜1 minute (no mixinginvolved). After the final wash step, the beads were resuspended in 15μl of elution buffer for 5 minutes at 70° C., followed by bead captureand the removal of the supernatant (up to 8 μl) for cDNA synthesisand/or purified RNA storage (−20° C.).

Real Time PCR

Two μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqManProbe (Applied Biosystems Cat #4326317E), 0.5 μl SERPINA1 TaqMan probe(Applied Biosystems cat # Hs00165475_ml) for Hep3B experiments or withcustom designed GAPDH and SERPINA1 taqman assays for PCH experiments and5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per wellin a 384 well plates (Roche cat #04887301001). Real time PCR was done ina Roche LC480 Real Time PCR system (Roche). Each duplex was tested in atleast two independent transfections with two biological replicates each,and each transfection was assayed in duplicate.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells. For freeuptake assays the data were normalized to PBS or GaINAc-1955 (highestconcentration used for experimental compounds) treated cells. IC₅₀s werecalculated using a 4 parameter fit model using XLFit and normalized tocells transfected with AD-1955 over the same dose range, or to its ownlowest dose.

The sense and antisense sequences of AD-1955 are: SENSE:5′-cuuAcGcuGAGuAcuucGAdTsdT-3′(SEQ ID NO: 33); and ANTISENSE:5′-UCGAAGuACUcAGCGuAAGdTsdT-3′(SEQ ID NO: 40).

The Taqman primers and probes used are as follows:

Cynomolgus Serpina1 and Gapdh TaqMan Primers and Probes:

Serpina1: Forward Primer: ACTAAGGTCTTCAGCAATGGG (SEQ ID NO:34); ReversePrimer: GCTTCAGTCCCTTTCTCATCG (SEQ ID NO:35); Taqman Probe:TGGTCAGCACAGCCTTATGCACG (SEQ ID NO:36) Gapdh: Forward Primer:GCATCCTGGGCTACACTGA (SEQ ID NO:37); Reverse Primer:TGGGTGTCGCTGTTGAAGTC(SEQ ID NO:38); Taqman Probe: CCAGGTGGTCTCCTCC (SEQID NO:39)

TABLE B Abbreviations of nucleotide monomers used in nucleic acidsequence representation. Ab- brevi- ation Nucleotide(s) AAdenosine-3′-phosphate Af 2′-fluoroadenosine-3′-phosphate Afs2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioateC cytidine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Cfs2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate Gguanosine-3′-phosphate Gf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioateUs uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate g2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate dT 2′-deoxythymidine dTs2′-deoxythymidine-3′-phosphorothioate dU 2′-deoxyuridine sphosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol Hyp-(GalNAc-alkyl)3 I inosine-3′-phosphate Is inosine-3′-phosphorothioate dI2′-deoxyriboinosine dIs 2′-deoxyinosine-3′-phosphorothioate Y342-hydroxymethyl-tetrahydrofurane-4-methoxy-3-phosphate (abasic 2′-OMefuranose) Y34s2-hydroxymethyl-tetrahydrofurane-4-methoxy-3-phosphorothioate (abasic2′-OMe furanose) P 5′-phosphate

Example 1. Synthesis of GalNAc-Conjugated Oligonucleotides

A series of siRNA duplexes spanning the sequence of Serpina1 mRNA weredesigned, synthesized, and conjugated with a trivalent GalNAc at the3-end of the sense strand using the techniques described above. Thesequences of these duplexes are shown in Table 1. These same sequenceswere also synthesized with various nucleotide modifications andconjugated with a trivalent GalNAc. The sequences of the modifiedduplexes are shown in Table 2.

TABLE 1 Serpina1 unmodified sequences Duplex Sense SEQ ID Position inAntisense SEQ ID Position in Name Oligo Name Sense Trans Seq NO:NM_000295.4 Oligo Name Antisense Trans Sequence NO: NM _000295.4AD-58681.1 A-119065.1 GUCCAACAGCACCAAUAUCUU  41 469-489 A-119066.1AAGAUAUUGGUGCUGUUGGACUG 129 467-489 AD-59084.1 A-119065.2GUCCAACAGCACCAAUAUCUU  42 469-489 A-119941.1 AAGAUAUUGGUGCUGUUGGACUG 130467-489 AD-59060.2 A-119933.1 UAAUGAUUGAACAAAAUACCA  43 1455-1475A-119934.1 UGGUAUUUUGUUCAAUCAUUAAG 131 1453-1475 AD-59060.1 A-119933.1UAAUGAUUGAACAAAAUACCA  44 1455-1475 A-119934.1 UGGUAUUUUGUUCAAUCAUUAAG132 1453-1475 AD-59054.2 A-119931.1 CUUCUUAAUGAUUGAACAAAA  45 1450-1470A-119932.1 UUUUGUUCAAUCAUUAAGAAGAC 133 1448-1470 AD-59054.1 A-119931.1CUUCUUAAUGAUUGAACAAAA  46 1450-1470 A-119932.1 UUUUGUUCAAUCAUUAAGAAGAC134 1448-1470 AD-59072.2 A-119937.1 AUUGAACAAAAUACCAAGUCU  47 1460-1480A-119938.1 AGACUUGGUAUUUUGUUCAAUCA 135 1458-1480 AD-59072.1 A-119937.1AUUGAACAAAAUACCAAGUCU  48 1460-1480 A-119938.1 AGACUUGGUAUUUUGUUCAAUCA136 1458-1480 AD-59048.2 A-119929.1 UUCUUAAUGAUUGAACAAAAU  49 1451-1471A-119930.1 AUUUUGUUCAAUCAUUAAGAAGA 137 1449-1471 AD-59048.1 A-119929.1UUCUUAAUGAUUGAACAAAAU  50 1451-1471 A-119930.1 AUUUUGUUCAAUCAUUAAGAAGA138 1449-1471 AD-59062.2 A-119964.1 CAAACCCUUUGUCUUCUUAAU  51 1438-1458A-119965.1 AUUAAGAAGACAAAGGGUUUGUU 139 1436-1458 AD-59062.1 A-119964.1CAAACCCUUUGUCUUCUUAAU  52 1438-1458 A-119965.1 AUUAAGAAGACAAAGGGUUUGUU140 1436-1458 AD-59078.2 A-119939.1 UGUCUUCUUAAUGAUUGAACA  53 1447-1467A-119940.1 UGUUCAAUCAUUAAGAAGACAAA 141 1445-1467 AD-59078.1 A-119939.1UGUCUUCUUAAUGAUUGAACA  54 1447-1467 A-119940.1 UGUUCAAUCAUUAAGAAGACAAA142 1445-1467 AD-59056.2 A-119962.1 CACCUGGAAAAUGAACUCACC  55 1121-1141A-119963.1 GGUGAGUUCAUUUUCCAGGUGCU 143 1119-1141 AD-59056.1 A-119962.1CACCUGGAAAAUGAACUCACC  56 1121-1141 A-119963.1 GGUGAGUUCAUUUUCCAGGUGCU144 1119-1141 AD-59091.2 A-119958.1 UUUUGCUCUGGUGAAUUACAU  57 880-900A-119959.1 AUGUAAUUCACCAGAGCAAAAAC 145 878-900 AD-59091.1 A-119958.1UUUUGCUCUGGUGAAUUACAU  58 880-900 A-119959.1 AUGUAAUUCACCAGAGCAAAAAC 146878-900 AD-59083.2 A-120018.1 ACCCUUUGUCUUCUUAAUGAU  59 1441-1461A-120019.1 AUCAUUAAGAAGACAAAGGGUUU 147 1439-1461 AD-59083.1 A-120018.1ACCCUUUGUCUUCUUAAUGAU  60 1441-1461 A-120019.1 AUCAUUAAGAAGACAAAGGGUUU148 1439-1461 AD-59073.2 A-119952.1 UUGAACAAAAUACCAAGUCUC  61 1461-1481A-119953.1 GAGACUUGGUAUUUUGUUCAAUC 149 1459-1481 AD-59073.1 A-119952.1UUGAACAAAAUACCAAGUCUC  62 1461-1481 A-119953.1 GAGACUUGGUAUUUUGUUCAAUC150 1459-1481 AD-59066.2 A-119935.1 GUUCAACAAACCCUUUGUCUU  63 1432-1452A-119936.1 AAGACAAAGGGUUUGUUGAACUU 151 1430-1452 AD-59066.1 A-119935.1GUUCAACAAACCCUUUGUCUU  64 1432-1452 A-119936.1 AAGACAAAGGGUUUGUUGAACUU152 1430-1452 AD-59059.2 A-120010.1 AAAUACCAAGUCUCCCCUCUU  65 1468-1488A-120011.1 AAGAGGGGAGACUUGGUAUUUUG 153 1466-1488 AD-59059.1 A-120010.1AAAUACCAAGUCUCCCCUCUU  66 1468-1488 A-120011.1 AAGAGGGGAGACUUGGUAUUUUG154 1466-1488 AD-59070.2 A-119998.1 UUUUUGCUCUGGUGAAUUACA  67 879-899A-119999.1 UGUAAUUCACCAGAGCAAAAACU 155 877-899 AD-59070.1 A-119998.1UUUUUGCUCUGGUGAAUUACA  68 879-899 A-119999.1 UGUAAUUCACCAGAGCAAAAACU 156877-899 AD-59063.2 A-119980.1 AGUUCAACAAACCCUUUGUCU  69 1431-1451A-119981.1 AGACAAAGGGUUUGUUGAACUUG 157 1429-1451 AD-59063.1 A-119980.1AGUUCAACAAACCCUUUGUCU  70 1431-1451 A-119981.1 AGACAAAGGGUUUGUUGAACUUG158 1429-1451 AD-59069.2 A-119982.1 AAUGAUUGAACAAAAUACCAA  71 1456-1476A-119983.1 UUGGUAUUUUGUUCAAUCAUUAA 159 1454-1476 AD-59069.1 A-119982.1AAUGAUUGAACAAAAUACCAA  72 1456-1476 A-119983.1 UUGGUAUUUUGUUCAAUCAUUAA160 1454-1476 AD-59082.2 A-120002.1 UACUGGAACCUAUGAUCUGAA  73 1216-1236A-120003.1 UUCAGAUCAUAGGUUCCAGUAAU 161 1214-1236 AD-59082.1 A-120002.1UACUGGAACCUAUGAUCUGAA  74 1216-1236 A-120003.1 UUCAGAUCAUAGGUUCCAGUAAU162 1214-1236 AD-59088.2 A-120004.1 ACAUUAAAGAAGGGUUGAGCU  75 1576-1596A-120005.1 AGCUCAACCCUUCUUUAAUGUCA 163 1574-1596 AD-59088.1 A-120004.1ACAUUAAAGAAGGGUUGAGCU  76 1576-1596 A-120005.1 AGCUCAACCCUUCUUUAAUGUCA164 1574-1596 AD-59080.2 A-119970.1 AAAAUUGUGGAUUUGGUCAAG  77 839-859A-119971.1 CUUGACCAAAUCCACAAUUUUCC 165 837-859 AD-59080.1 A-119970.1AAAAUUGUGGAUUUGGUCAAG  78 839-859 A-119971.1 CUUGACCAAAUCCACAAUUUUCC 166837-859 AD-59058.2 A-119994.1 AUUACUGGAACCUAUGAUCUG  79 1214-1234A-119995.1 CAGAUCAUAGGUUCCAGUAAUGG 167 1212-1234 AD-59058.1 A-119994.1AUUACUGGAACCUAUGAUCUG  80 1214-1234 A-119995.1 CAGAUCAUAGGUUCCAGUAAUGG168 1212-1234 AD-59090.2 A-119942.1 CACAGUUUUUGCUCUGGUGAA  81 874-894A-119943.1 UUCACCAGAGCAAAAACUGUGUC 169 872-894 AD-59090.1 A-119942.1CACAGUUUUUGCUCUGGUGAA  82 874-894 A-119943.1 UUCACCAGAGCAAAAACUGUGUC 170872-894 AD-59057.2 A-119978.1 UUAAAGAAGGGUUGAGCUGGU  83 1579-1599A-119979.1 ACCAGCUCAACCCUUCUUUAAUG 171 1577-1599 AD-59057.1 A-119978.1UUAAAGAAGGGUUGAGCUGGU  84 1579-1599 A-119979.1 ACCAGCUCAACCCUUCUUUAAUG172 1577-1599 AD-59051.2 A-119976.1 AGUGAGCAUCGCUACAGCCUU  85 499-519A-119977.1 AAGGCUGUAGCGAUGCUCACUGG 173 497-519 AD-59051.1 A-119976.1AGUGAGCAUCGCUACAGCCUU  86 499-519 A-119977.1 AAGGCUGUAGCGAUGCUCACUGG 174497-519 AD-59065.2 A-120012.1 AAGGAGCUUGACAGAGACACA  87 857-877A-120013.1 UGUGUCUCUGUCAAGCUCCUUGA 175 855-877 AD-59065.1 A-120012.1AAGGAGCUUGACAGAGACACA  88 857-877 A-120013.1 UGUGUCUCUGUCAAGCUCCUUGA 176855-877 AD-59087.2 A-119988.1 GUGGAUAAGUUUUUGGAGGAU  89 716-736A-119989.1 AUCCUCCAAAAACUUAUCCACUA 177 714-736 AD-59087.1 A-119988.1GUGGAUAAGUUUUUGGAGGAU  90 716-736 A-119989.1 AUCCUCCAAAAACUUAUCCACUA 178714-736 AD-59075.2 A-119984.1 GAUUGAACAAAAUACCAAGUC  91 1459-1479A-119985.1 GACUUGGUAUUUUGUUCAAUCAU 179 1457-1479 AD-59075.1 A-119984.1GAUUGAACAAAAUACCAAGUC  92 1459-1479 A-119985.1 GACUUGGUAUUUUGUUCAAUCAU180 1457-1479 AD-59092.2 A-119974.1 GCUCUCCAAGGCCGUGCAUAA  93 1321-1341A-119975.1 UUAUGCACGGCCUUGGAGAGCUU 181 1319-1341 AD-59092.1 A-119974.1GCUCUCCAAGGCCGUGCAUAA  94 1321-1341 A-119975.1 UUAUGCACGGCCUUGGAGAGCUU182 1319-1341 AD-59081.2 A-119986.1 ACCUGGAAAAUGAACUCACCC  95 1122-1142A-119987.1 GGGUGAGUUCAUUUUCCAGGUGC 183 1120-1142 AD-59081.1 A-119986.1ACCUGGAAAAUGAACUCACCC  96 1122-1142 A-119987.1 GGGUGAGUUCAUUUUCCAGGUGC184 1120-1142 AD-59064.2 A-119996.1 GGGACCAAGGCUGACACUCAC  97 536-556A-119997.1 GUGAGUGUCAGCCUUGGUCCCCA 185 534-556 AD-59064.1 A-119996.1GGGACCAAGGCUGACACUCAC  98 536-556 A-119997.1 GUGAGUGUCAGCCUUGGUCCCCA 186534-556 AD-59052.2 A-119992.1 GCCAUGUUUUUAGAGGCCAUA  99 1385-1405A-119993.1 UAUGGCCUCUAAAAACAUGGCCC 187 1383-1405 AD-59052.1 A-119992.1GCCAUGUUUUUAGAGGCCAUA 100 1385-1405 A-119993.1 UAUGGCCUCUAAAAACAUGGCCC188 1383-1405 AD-59076.2 A-120000.1 CCUGGAAAAUGAACUCACCCA 101 1123-1143A-120001.1 UGGGUGAGUUCAUUUUCCAGGUG 189 1121-1143 AD-59076.1 A-120000.1CCUGGAAAAUGAACUCACCCA 102 1123-1143 A-120001.1 UGGGUGAGUUCAUUUUCCAGGUG190 1121-1143 AD-59068.2 A-119966.1 AAGAGGCCAAGAAACAGAUCA 103 789-809A-119967.1 UGAUCUGUUUCUUGGCCUCUUCG 191 787-809 AD-59068.1 A-119966.1AAGAGGCCAAGAAACAGAUCA 104 789-809 A-119967.1 UGAUCUGUUUCUUGGCCUCUUCG 192787-809 AD-59089.2 A-120020.1 GGCAAAUGGGAGAGACCCUUU 105 911-931A-120021.1 AAAGGGUCUCUCCCAUUUGCCUU 193 909-931 AD-59089.1 A-120020.1GGCAAAUGGGAGAGACCCUUU 106 911-931 A-120021.1 AAAGGGUCUCUCCCAUUUGCCUU 194909-931 AD-59093.2 A-119990.1 UGGGAAAAGUGGUGAAUCCCA 107 1491-1511A-119991.1 UGGGAUUCACCACUUUUCCCAUG 195 1489-1511 AD-59093.1 A-119990.1UGGGAAAAGUGGUGAAUCCCA 108 1491-1511 A-119991.1 UGGGAUUCACCACUUUUCCCAUG196 1489-1511 AD-59061.2 A-119948.1 GGGGACCAAGGCUGACACUCA 109 535-555A-119949.1 UGAGUGUCAGCCUUGGUCCCCAG 197 533-555 AD-59061.1 A-119948.1GGGGACCAAGGCUGACACUCA 110 535-555 A-119949.1 UGAGUGUCAGCCUUGGUCCCCAG 198533-555 AD-59074.2 A-119968.1 GACAUUAAAGAAGGGUUGAGC 111 1575-1595A-119969.1 GCUCAACCCUUCUUUAAUGUCAU 199 1573-1595 AD-59074.1 A-119968.1GACAUUAAAGAAGGGUUGAGC 112 1575-1595 A-119969.1 GCUCAACCCUUCUUUAAUGUCAU200 1573-1595 AD-59079.2 A-119954.1 GGCCAUGUUUUUAGAGGCCAU 113 1384-1404A-119955.1 AUGGCCUCUAAAAACAUGGCCCC 201 1382-1404 AD-59079.1 A-119954.1GGCCAUGUUUUUAGAGGCCAU 114 1384-1404 A-119955.1 AUGGCCUCUAAAAACAUGGCCCC202 1382-1404 AD-59071.2 A-120014.1 UUCCUGCCUGAUGAGGGGAAA 115 1094-1114A-120015.1 UUUCCCCUCAUCAGGCAGGAAGA 203 1092-1114 AD-59071.1 A-120014.1UUCCUGCCUGAUGAGGGGAAA 116 1094-1114 A-120015.1 UUUCCCCUCAUCAGGCAGGAAGA204 1092-1114 AD-59086.2 A-119972.1 CUCUCCAAGGCCGUGCAUAAG 117 1322-1342A-119973.1 CUUAUGCACGGCCUUGGAGAGCU 205 1320-1342 AD-59086.1 A-119972.1CUCUCCAAGGCCGUGCAUAAG 118 1322-1342 A-119973.1 CUUAUGCACGGCCUUGGAGAGCU206 1320-1342 AD-59094.2 A-120006.1 AGCUCUCCAAGGCCGUGCAUA 119 1320-1340A-120007.1 UAUGCACGGCCUUGGAGAGCUUC 207 1318-1340 AD-59094.1 A-120006.1AGCUCUCCAAGGCCGUGCAUA 120 1320-1340 A-120007.1 UAUGCACGGCCUUGGAGAGCUUC208 1318-1340 AD-59085.2 A-119956.1 UCCUGGAGGGCCUGAAUUUCA 121 564-584A-119957.1 UGAAAUUCAGGCCCUCCAGGAUU 209 562-584 AD-59085.1 A-119956.1UCCUGGAGGGCCUGAAUUUCA 122 564-584 A-119957.1 UGAAAUUCAGGCCCUCCAGGAUU 210562-584 AD-59067.2 A-119950.1 UUGGUCAAGGAGCUUGACAGA 123 851-871A-119951.1 UCUGUCAAGCUCCUUGACCAAAU 211 849-871 AD-59067.1 A-119950.1UUGGUCAAGGAGCUUGACAGA 124 851-871 A-119951.1 UCUGUCAAGCUCCUUGACCAAAU 212849-871 AD-59053.2 A-120008.1 UUUGGUCAAGGAGCUUGACAG 125 850-870A-120009.1 CUGUCAAGCUCCUUGACCAAAUC 213 848-870 AD-59053.1 A-120008.1UUUGGUCAAGGAGCUUGACAG 126 850-870 A-120009.1 CUGUCAAGCUCCUUGACCAAAUC 214848-870 AD-59077.2 A-120016.1 UCCCCAGUGAGCAUCGCUACA 127 494-514A-120017.1 UGUAGCGAUGCUCACUGGGGAGA 215 492-514 AD-59077.1 A-120016.1UCCCCAGUGAGCAUCGCUACA 128 494-514 A-120017.1 UGUAGCGAUGCUCACUGGGGAGA 216492-514

TABLE 2 Serpina1-modified sequences Duplex Sense SEQ Antisense SEQ IDName Oligo Name Sense Oligo Sequence ID NO: Oligo NameAntisense Oligo Sequence NO: AD-58681.1 A-119065.1GfsusCfcAfaCfaGfCfAfcCfaAfuAfuCfuUfL96 217 A-119066.1asAfsgAfuAfuUfgGfugcUfgUfuGfgAfcsUfsg 305 AD-59084.1 A-119065.2GfsusCfcAfaCfaGfCfAfcCfaAfuAfuCfuUfL96 218 A-119941.1asAfsgAfuAfuUfgGfugcUfgUfuGfgAfcsusg 306 AD-59060.2 A-119933.1UfsasAfuGfaUfuGfAfAfcAfaAfaUfaCfcAfL96 219 A-119934.1usGfsgUfaUfuUfuGfuucAfaUfcAfuUfasasg 307 AD-59060.1 A-119933.1UfsasAfuGfaUfuGfAfAfcAfaAfaUfaCfcAfL96 220 A-119934.1usGfsgUfaUfuUfuGfuucAfaUfcAfuUfasasg 308 AD-59054.2 A-119931.1CfsusUfcUfuAfaUfGfAfuUfgAfaCfaAfaAfL96 221 A-119932.1usUfsuUfgUfuCfaAfucaUfuAfaGfaAfgsasc 309 AD-59054.1 A-119931.1CfsusUfcUfuAfaUfGfAfuUfgAfaCfaAfaAfL96 222 A-119932.1usUfsuUfgUfuCfaAfucaUfuAfaGfaAfgsasc 310 AD-59072.2 A-119937.1AfsusUfgAfaCfaAfAfAfuAfcCfaAfgUfcUfL96 223 A-119938.1asGfsaCfuUfgGfuAfuuuUfgUfuCfaAfuscsa 311 AD-59072.1 A-119937.1AfsusUfgAfaCfaAfAfAfuAfcCfaAfgUfcUfL96 224 A-119938.1asGfsaCfuUfgGfuAfuuuUfgUfuCfaAfuscsa 312 AD-59048.2 A-119929.1UfsusCfuUfaAfuGfAfUfuGfaAfcAfaAfaUfL96 225 A-119930.1asUfsuUfuGfuUfcAfaucAfuUfaAfgAfasgsa 313 AD-59048.1 A-119929.1UfsusCfuUfaAfuGfAfUfuGfaAfcAfaAfaUfL96 226 A-119930.1asUfsuUfuGfuUfcAfaucAfuUfaAfgAfasgsa 314 AD-59062.2 A-119964.1CfsasAfaCfcCfuUfUfGfuCfuUfcUfuAfaUfL96 227 A-119965.1asUfsuAfaGfaAfgAfcaaAfgGfgUfuUfgsusu 315 AD-59062.1 A-119964.1CfsasAfaCfcCfuUfUfGfuCfuUfcUfuAfaUfL96 228 A-119965.1asUfsuAfaGfaAfgAfcaaAfgGfgUfuUfgsusu 316 AD-59078.2 A-119939.1UfsgsUfcUfuCfuUfAfAfuGfaUfuGfaAfcAfL96 229 A-119940.1usGfsuUfcAfaUfcAfuuaAfgAfaGfaCfasasa 317 AD-59078.1 A-119939.1UfsgsUfcUfuCfuUfAfAfuGfaUfuGfaAfcAfL96 230 A-119940.1usGfsuUfcAfaUfcAfuuaAfgAfaGfaCfasasa 318 AD-59056.2 A-119962.1CfsasCfcUfgGfaAfAfAfuGfaAfcUfcAfcCfL96 231 A-119963.1gsGfsuGfaGfuUfcAfuuuUfcCfaGfgUfgscsu 319 AD-59056.1 A-119962.1CfsasCfcUfgGfaAfAfAfuGfaAfcUfcAfcCfL96 232 A-119963.1gsGfsuGfaGfuUfcAfuuuUfcCfaGfgUfgscsu 320 AD-59091.2 A-119958.1UfsusUfuGfcUfcUfGfGfuGfaAfuUfaCfaUfL96 233 A-119959.1asUfsgUfaAfuUfcAfccaGfaGfcAfaAfasasc 321 AD-59091.1 A-119958.1UfsusUfuGfcUfcUfGfGfuGfaAfuUfaCfaUfL96 234 A-119959.1asUfsgUfaAfuUfcAfccaGfaGfcAfaAfasasc 322 AD-59083.2 A-120018.1AfscsCfcUfuUfgUfCfUfuCfuUfaAfuGfaUfL96 235 A-120019.1asUfscAfuUfaAfgAfagaCfaAfaGfgGfususu 323 AD-59083.1 A-120018.1AfscsCfcUfuUfgUfCfUfuCfuUfaAfuGfaUfL96 236 A-120019.1asUfscAfuUfaAfgAfagaCfaAfaGfgGfususu 324 AD-59073.2 A-119952.1UfsusGfaAfcAfaAfAfUfaCfcAfaGfuCfuCfL96 237 A-119953.1gsAfsgAfcUfuGfgUfauuUfuGfuUfcAfasusc 325 AD-59073.1 A-119952.1UfsusGfaAfcAfaAfAfUfaCfcAfaGfuCfuCfL96 238 A-119953.1gsAfsgAfcUfuGfgUfauuUfuGfuUfcAfasusc 326 AD-59066.2 A-119935.1GfsusUfcAfaCfaAfAfCfcCfuUfuGfuCfuUfL96 239 A-119936.1asAfsgAfcAfaAfgGfguuUfgUfuGfaAfcsusu 327 AD-59066.1 A-119935.1GfsusUfcAfaCfaAfAfCfcCfuUfuGfuCfuUfL96 240 A-119936.1asAfsgAfcAfaAfgGfguuUfgUfuGfaAfcsusu 328 AD-59059.2 A-120010.1AfsasAfuAfcCfaAfGfUfcUfcCfcCfuCfuUfL96 241 A-120011.1asAfsgAfgGfgGfaGfacuUfgGfuAfuUfususg 329 AD-59059.1 A-120010.1AfsasAfuAfcCfaAfGfUfcUfcCfcCfuCfuUfL96 242 A-120011.1asAfsgAfgGfgGfaGfacuUfgGfuAfuUfususg 330 AD-59070.2 A-119998.1UfsusUfuUfgCfuCfUfGfgUfgAfaUfuAfcAfL96 243 A-119999.1usGfsuAfaUfuCfaCfcagAfgCfaAfaAfascsu 331 AD-59070.1 A-119998.1UfsusUfuUfgCfuCfUfGfgUfgAfaUfuAfcAfL96 244 A-119999.1usGfsuAfaUfuCfaCfcagAfgCfaAfaAfascsu 332 AD-59063.2 A-119980.1AfsgsUfuCfaAfcAfAfAfcCfcUfuUfgUfcUfL96 245 A-119981.1asGfsaCfaAfaGfgGfuuuGfuUfgAfaCfususg 333 AD-59063.1 A-119980.1AfsgsUfuCfaAfcAfAfAfcCfcUfuUfgUfcUfL96 246 A-119981.1asGfsaCfaAfaGfgGfuuuGfuUfgAfaCfususg 334 AD-59069.2 A-119982.1AfsasUfgAfuUfgAfAfCfaAfaAfuAfcCfaAfL96 247 A-119983.1usUfsgGfuAfuUfuUfguuCfaAfuCfaUfusasa 335 AD-59069.1 A-119982.1AfsasUfgAfuUfgAfAfCfaAfaAfuAfcCfaAfL96 248 A-119983.1usUfsgGfuAfuUfuUfguuCfaAfuCfaUfusasa 336 AD-59082.2 A-120002.1UfsasCfuGfgAfaCfCfUfaUfgAfuCfuGfaAfL96 249 A-120003.1usUfscAfgAfuCfaUfaggUfuCfcAfgUfasasu 337 AD-59082.1 A-120002.1UfsasCfuGfgAfaCfCfUfaUfgAfuCfuGfaAfL96 250 A-120003.1usUfscAfgAfuCfaUfaggUfuCfcAfgUfasasu 338 AD-59088.2 A-120004.1AfscsAfuUfaAfaGfAfAfgGfgUfuGfaGfcUfL96 251 A-120005.1asGfscUfcAfaCfcCfuucUfuUfaAfuGfuscsa 339 AD-59088.1 A-120004.1AfscsAfuUfaAfaGfAfAfgGfgUfuGfaGfcUfL96 252 A-120005.1asGfscUfcAfaCfcCfuucUfuUfaAfuGfuscsa 340 AD-59080.2 A-119970.1AfsasAfaUfuGfuGfGfAfuUfuGfgUfcAfaGfL96 253 A-119971.1csUfsuGfaCfcAfaAfuccAfcAfaUfuUfuscsc 341 AD-59080.1 A-119970.1AfsasAfaUfuGfuGfGfAfuUfuGfgUfcAfaGfL96 254 A-119971.1csUfsuGfaCfcAfaAfuccAfcAfaUfuUfuscsc 342 AD-59058.2 A-119994.1AfsusUfaCfuGfgAfAfCfcUfaUfgAfuCfuGfL96 255 A-119995.1csAfsgAfuCfaUfaGfguuCfcAfgUfaAfusgsg 343 AD-59058.1 A-119994.1AfsusUfaCfuGfgAfAfCfcUfaUfgAfuCfuGfL96 256 A-119995.1csAfsgAfuCfaUfaGfguuCfcAfgUfaAfusgsg 344 AD-59090.2 A-119942.1CfsasCfaGfuUfuUfUfGfcUfcUfgGfuGfaAfL96 257 A-119943.1usUfscAfcCfaGfaGfcaaAfaAfcUfgUfgsusc 345 AD-59090.1 A-119942.1CfsasCfaGfuUfuUfUfGfcUfcUfgGfuGfaAfL96 258 A-119943.1usUfscAfcCfaGfaGfcaaAfaAfcUfgUfgsusc 346 AD-59057.2 A-119978.1UfsusAfaAfgAfaGfGfGfuUfgAfgCfuGfgUfL96 259 A-119979.1asCfscAfgCfuCfaAfcccUfuCfuUfuAfasusg 347 AD-59057.1 A-119978.1UfsusAfaAfgAfaGfGfGfuUfgAfgCfuGfgUfL96 260 A-119979.1asCfscAfgCfuCfaAfcccUfuCfuUfuAfasusg 348 AD-59051.2 A-119976.1AfsgsUfgAfgCfaUfCfGfcUfaCfaGfcCfuUfL96 261 A-119977.1asAfsgGfcUfgUfaGfcgaUfgCfuCfaCfusgsg 349 AD-59051.1 A-119976.1AfsgsUfgAfgCfaUfCfGfcUfaCfaGfcCfuUfL96 262 A-119977.1asAfsgGfcUfgUfaGfcgaUfgCfuCfaCfusgsg 350 AD-59065.2 A-120012.1AfsasGfgAfgCfuUfGfAfcAfgAfgAfcAfcAfL96 263 A-120013.1usGfsuGfuCfuCfuGfucaAfgCfuCfcUfusgsa 351 AD-59065.1 A-120012.1AfsasGfgAfgCfuUfGfAfcAfgAfgAfcAfcAfL96 264 A-120013.1usGfsuGfuCfuCfuGfucaAfgCfuCfcUfusgsa 352 AD-59087.2 A-119988.1GfsusGfgAfuAfaGfUfUfuUfuGfgAfgGfaUfL96 265 A-119989.1asUfscCfuCfcAfaAfaacUfuAfuCfcAfcsusa 353 AD-59087.1 A-119988.1GfsusGfgAfuAfaGfUfUfuUfuGfgAfgGfaUfL96 266 A-119989.1asUfscCfuCfcAfaAfaacUfuAfuCfcAfcsusa 354 AD-59075.2 A-119984.1GfsasUfuGfaAfcAfAfAfaUfaCfcAfaGfuCfL96 267 A-119985.1gsAfscUfuGfgUfaUfuuuGfuUfcAfaUfcsasu 355 AD-59075.1 A-119984.1GfsasUfuGfaAfcAfAfAfaUfaCfcAfaGfuCfL96 268 A-119985.1gsAfscUfuGfgUfaUfuuuGfuUfcAfaUfcsasu 356 AD-59092.2 A-119974.1GfscsUfcUfcCfaAfGfGfcCfgUfgCfaUfaAfL96 269 A-119975.1usUfsaUfgCfaCfgGfccuUfgGfaGfaGfcsusu 357 AD-59092.1 A-119974.1GfscsUfcUfcCfaAfGfGfcCfgUfgCfaUfaAfL96 270 A-119975.1usUfsaUfgCfaCfgGfccuUfgGfaGfaGfcsusu 358 AD-59081.2 A-119986.1AfscsCfuGfgAfaAfAfUfgAfaCfuCfaCfcCfL96 271 A-119987.1gsGfsgUfgAfgUfuCfauuUfuCfcAfgGfusgsc 359 AD-59081.1 A-119986.1AfscsCfuGfgAfaAfAfUfgAfaCfuCfaCfcCfL96 272 A-119987.1gsGfsgUfgAfgUfuCfauuUfuCfcAfgGfusgsc 360 AD-59064.2 A-119996.1GfsgsGfaCfcAfaGfGfCfuGfaCfaCfuCfaCfL96 273 A-119997.1gsUfsgAfgUfgUfcAfgccUfuGfgUfcCfcscsa 361 AD-59064.1 A-119996.1GfsgsGfaCfcAfaGfGfCfuGfaCfaCfuCfaCfL96 274 A-119997.1gsUfsgAfgUfgUfcAfgccUfuGfgUfcCfcscsa 362 AD-59052.2 A-119992.1GfscsCfaUfgUfuUfUfUfaGfaGfgCfcAfuAfL96 275 A-119993.1usAfsuGfgCfcUfcUfaaaAfaCfaUfgGfcscsc 363 AD-59052.1 A-119992.1GfscsCfaUfgUfuUfUfUfaGfaGfgCfcAfuAfL96 276 A-119993.1usAfsuGfgCfcUfcUfaaaAfaCfaUfgGfcscsc 364 AD-59076.2 A-120000.1CfscsUfgGfaAfaAfUfGfaAfcUfcAfcCfcAfL96 277 A-120001.1usGfsgGfuGfaGfuUfcauUfuUfcCfaGfgsusg 365 AD-59076.1 A-120000.1CfscsUfgGfaAfaAfUfGfaAfcUfcAfcCfcAfL96 278 A-120001.1usGfsgGfuGfaGfuUfcauUfuUfcCfaGfgsusg 366 AD-59068.2 A-119966.1AfsasGfaGfgCfcAfAfGfaAfaCfaGfaUfcAfL96 279 A-119967.1usGfsaUfcUfgUfuUfcuuGfgCfcUfcUfuscsg 367 AD-59068.1 A-119966.1AfsasGfaGfgCfcAfAfGfaAfaCfaGfaUfcAfL96 280 A-119967.1usGfsaUfcUfgUfuUfcuuGfgCfcUfcUfuscsg 368 AD-59089.2 A-120020.1GfsgsCfaAfaUfgGfGfAfgAfgAfcCfcUfuUfL96 281 A-120021.1asAfsaGfgGfuCfuCfuccCfaUfuUfgCfcsusu 369 AD-59089.1 A-120020.1GfsgsCfaAfaUfgGfGfAfgAfgAfcCfcUfuUfL96 282 A-120021.1asAfsaGfgGfuCfuCfuccCfaUfuUfgCfcsusu 370 AD-59093.2 A-119990.1UfsgsGfgAfaAfaGfUfGfgUfgAfaUfcCfcAfL96 283 A-119991.1usGfsgGfaUfuCfaCfcacUfuUfuCfcCfasusg 371 AD-59093.1 A-119990.1UfsgsGfgAfaAfaGfUfGfgUfgAfaUfcCfcAfL96 284 A-119991.1usGfsgGfaUfuCfaCfcacUfuUfuCfcCfasusg 372 AD-59061.2 A-119948.1GfsgsGfgAfcCfaAfGfGfcUfgAfcAfcUfcAfL96 285 A-119949.1usGfsaGfuGfuCfaGfccuUfgGfuCfcCfcsasg 373 AD-59061.1 A-119948.1GfsgsGfgAfcCfaAfGfGfcUfgAfcAfcUfcAfL96 286 A-119949.1usGfsaGfuGfuCfaGfccuUfgGfuCfcCfcsasg 374 AD-59074.2 A-119968.1GfsasCfaUfuAfaAfGfAfaGfgGfuUfgAfgCfL96 287 A-119969.1gsCfsuCfaAfcCfcUfucuUfuAfaUfgUfcsasu 375 AD-59074.1 A-119968.1GfsasCfaUfuAfaAfGfAfaGfgGfuUfgAfgCfL96 288 A-119969.1gsCfsuCfaAfcCfcUfucuUfuAfaUfgUfcsasu 376 AD-59079.2 A-119954.1GfsgsCfcAfuGfuUfUfUfuAfgAfgGfcCfaUfL96 289 A-119955.1asUfsgGfcCfuCfuAfaaaAfcAfuGfgCfcscsc 377 AD-59079.1 A-119954.1GfsgsCfcAfuGfuUfUfUfuAfgAfgGfcCfaUfL96 290 A-119955.1asUfsgGfcCfuCfuAfaaaAfcAfuGfgCfcscsc 378 AD-59071.2 A-120014.1UfsusCfcUfgCfcUfGfAfuGfaGfgGfgAfaAfL96 291 A-120015.1usUfsuCfcCfcUfcAfucaGfgCfaGfgAfasgsa 379 AD-59071.1 A-120014.1UfsusCfcUfgCfcUfGfAfuGfaGfgGfgAfaAfL96 292 A-120015.1usUfsuCfcCfcUfcAfucaGfgCfaGfgAfasgsa 380 AD-59086.2 A-119972.1CfsusCfuCfcAfaGfGfCfcGfuGfcAfuAfaGfL96 293 A-119973.1csUfsuAfuGfcAfcGfgccUfuGfgAfgAfgscsu 381 AD-59086.1 A-119972.1CfsusCfuCfcAfaGfGfCfcGfuGfcAfuAfaGfL96 294 A-119973.1csUfsuAfuGfcAfcGfgccUfuGfgAfgAfgscsu 382 AD-59094.2 A-120006.1AfsgsCfuCfuCfcAfAfGfgCfcGfuGfcAfuAfL96 295 A-120007.1usAfsuGfcAfcGfgCfcuuGfgAfgAfgCfususc 383 AD-59094.1 A-120006.1AfsgsCfuCfuCfcAfAfGfgCfcGfuGfcAfuAfL96 296 A-120007.1usAfsuGfcAfcGfgCfcuuGfgAfgAfgCfususc 384 AD-59085.2 A-119956.1UfscsCfuGfgAfgGfGfCfcUfgAfaUfuUfcAfL96 297 A-119957.1usGfsaAfaUfuCfaGfgccCfuCfcAfgGfasusu 385 AD-59085.1 A-119956.1UfscsCfuGfgAfgGfGfCfcUfgAfaUfuUfcAfL96 298 A-119957.1usGfsaAfaUfuCfaGfgccCfuCfcAfgGfasusu 386 AD-59067.2 A-119950.1UfsusGfgUfcAfaGfGfAfgCfuUfgAfcAfgAfL96 299 A-119951.1usCfsuGfuCfaAfgCfuccUfuGfaCfcAfasasu 387 AD-59067.1 A-119950.1UfsusGfgUfcAfaGfGfAfgCfuUfgAfcAfgAfL96 300 A-119951.1usCfsuGfuCfaAfgCfuccUfuGfaCfcAfasasu 388 AD-59053.2 A-120008.1UfsusUfgGfuCfaAfGfGfaGfcUfuGfaCfaGfL96 301 A-120009.1csUfsgUfcAfaGfcUfccuUfgAfcCfaAfasusc 389 AD-59053.1 A-120008.1UfsusUfgGfuCfaAfGfGfaGfcUfuGfaCfaGfL96 302 A-120009.1csUfsgUfcAfaGfcUfccuUfgAfcCfaAfasusc 390 AD-59077.2 A-120016.1UfscsCfcCfaGfuGfAfGfcAfuCfgCfuAfcAfL96 303 A-120017.1usGfsuAfgCfgAfuGfcucAfcUfgGfgGfasgsa 391 AD-59077.1 A-120016.1UfscsCfcCfaGfuGfAfGfcAfuCfgCfuAfcAfL96 304 A-120017.1usGfsuAfgCfgAfuGfcucAfcUfgGfgGfasgsa 392

Example 2. In Vitro and In Vivo Screening

A subset of these duplexes was evaluated for efficacy in single doseassays as described above. Table 3 shows the results of a single dosescreen in primary mouse hepatocytes (Hep3b) transfected with theindicated GalNAC conjugated modified iRNAs and the results of a singledose free uptake screen in primary Cynomolgus hepatocytes (PCH) with theindicated GalNAC conjugated modified iRNAs. Data are expressed asfraction of message remaining relative to cells treated with AD-1955, anon-targeting control for Hep3B experiments, or relative to naïve cellsfor PCH experiments.

TABLE 3 Serpina1 efficacy screen by free uptake in primary Hep3b cellsand in primary Cynomolgous monkey hepatocytes (PCH). Transfection(Hep3b) Free Uptake (PCH) 10 nM 0.1 nM 10 nM 500 nM Avg SD Avg SD Avg SDAvg SD AD-58681 2.7 0.8 4.2 0.5 72.7 9.8 42.1 4.6 AD-59084 2.1 0.2 6.20.6 74.5 10.1 54.2 13.3 AD-59060 1.2 0.4 6.5 0.3 87.4 8.7 69.5 4.5AD-59054 2.2 1.4 7.2 0.7 59.1 10.8 50.3 5.0 AD-59072 1.3 0.3 7.7 0.287.6 6.4 86.2 9.9 AD-59048 1.1 0.4 8.1 0.4 72.9 19.5 46.4 5.8 AD-590621.4 0.0 9.2 0.6 77.9 11.6 64.9 11.0 AD-59078 1.8 0.0 12.1 0.2 89.2 9.371.1 3.2 AD-59056 1.8 0.1 20.2 1.8 88.9 13.4 83.7 8.5 AD-59091 3.8 0.526.6 4.1 89.7 15.0 75.6 7.5 AD-59083 2.3 0.6 27.2 2.5 94.5 9.1 74.5 11.9AD-59073 3.7 0.7 27.3 2.3 101.5 15.7 85.1 18.9 AD-59066 5.9 1.7 31.5 3.4106.2 25.3 28.2 27.1 AD-59059 2.9 0.7 32.9 3.4 101.3 10.4 84.9 18.0AD-59070 7.4 1.0 33.9 6.6 87.5 9.3 80.1 13.2 AD-59063 3.0 0.3 35.0 3.999.3 4.9 91.1 7.9 AD-59069 5.6 0.5 39.6 3.5 90.5 19.6 100.4 7.3 AD-590825.0 2.3 41.3 1.8 89.2 27.3 87.8 3.9 AD-59088 5.2 0.2 41.5 2.1 96.4 17.196.2 18.2 AD-59080 8.2 1.8 41.8 2.1 94.3 4.9 93.4 15.0 AD-59058 6.4 0.743.9 0.3 112.1 12.7 92.5 8.6 AD-59090 5.8 0.5 44.8 0.8 119.3 14.6 100.226.7 AD-59057 6.2 0.3 47.5 0.9 95.2 7.8 76.1 5.8 AD-59051 7.0 0.3 52.24.4 89.4 2.8 82.0 13.6 AD-59065 12.7 1.4 60.1 4.4 94.1 9.7 90.6 5.9AD-59087 7.7 1.0 62.1 4.7 92.3 6.8 72.6 10.4 AD-59075 9.3 2.3 62.9 2.0101.7 10.6 99.0 18.8 AD-59092 14.6 4.0 65.5 1.7 87.4 17.3 94.1 21.2AD-59081 10.9 2.3 68.2 2.4 115.1 18.4 106.1 11.8 AD-59064 11.0 0.1 71.64.5 91.3 14.7 87.2 10.3 AD-59052 21.8 2.6 78.6 2.4 99.9 9.2 88.9 17.5AD-59076 14.5 4.2 79.4 1.5 84.9 27.2 101.7 10.8 AD-59068 48.1 1.6 81.82.5 100.2 19.7 107.1 25.8 AD-59089 30.4 0.6 82.6 9.0 87.3 11.9 89.1 3.7AD-59093 23.5 0.2 85.2 5.4 72.1 48.5 103.0 13.2 AD-59061 38.1 2.2 86.54.4 100.3 13.3 102.3 9.0 AD-59074 38.9 5.4 86.6 3.0 106.5 10.3 100.614.7 AD-59079 45.1 0.8 87.6 4.8 100.5 17.4 92.1 33.3 AD-59071 58.6 1.096.2 7.1 82.3 25.8 110.7 2.2 AD-59086 78.3 1.1 96.3 4.1 93.1 7.3 97.117.0 AD-59094 96.6 2.7 102.1 0.8 75.2 52.7 76.9 7.9 AD-59085 99.3 3.7102.5 4.4 94.1 10.0 102.4 16.3 AD-59067 88.7 0.8 103.7 0.9 118.5 17.2108.9 30.3 AD-59053 98.5 4.7 103.7 1.9 98.7 14.8 96.4 8.1 AD-59077 100.58.2 104.8 1.6 88.0 32.5 88.1 4.1

The IC₅₀ values for selected duplexes by transfection in primaryHep3Bare shown in Table 4.

TABLE 4 Serpinal IC₅₀ values for selected duplexes by transfection inthe Hep3B human cell line. IC50 Duplex (nM) AD-58681 0.031 AD-590540.128 AD-59062 0.130 AD-59084 0.143 AD-59048 0.146 AD-59072 0.197AD-59056 0.408 AD-59078 0.600 AD-59066 0.819 AD-59060 1.883

A subset of these duplexes was evaluated for in vivo efficacy intransgenic mice expressing the Z-AAT form of human Serpina1 (see, e.g.,Dycaico, et al. (1988) Science 242:1409-12; Carlson, et al. (1989) JClin Invest 83:1183-90; Perfumo, et al. (1994) Ann Hum Genet. 58:305-20.This is an established model of AAT-deficiency associated liver disease.Briefly, transgenic mice were injected subcutaneously with a single 20mg/kg dose of the iRNAs listed in Table 5 at Day 0. Serum was collectedat Days −10, −5, 0, 3, 5, 7, 10, and 17 and the amount of circulatingSerpina1 protein was determined using a human-specific ELISA assay. Theresults of these analyses are depicted in FIG. 1. As indicated in FIG.1, AD-58681-6PS was the most effective in reducing serum Serpina1protein levels in these mice.

TABLE 5 AD-54330.2 A-111587.3 sense GfuCfcAfaCfaGfCfAfcCfaAfuAfuCfuUfL96(SEQ ID NO: 393) A-111588.3 antisenseaAfgAfuAfuUfgGfugcUfgUfuGfgAfcsUfsg (SEQ ID NO: 394) AD-58681.1A-119065.1 sense GfsusCfcAfaCfaGfCfAfcCfaAfuAfuCfuUfL96 (SEQ ID NO: 395)A-119066.1 antisense asAfsgAfuAfuUfgGfugcUfgUfuGfgAfcsUfsg(SEQ ID NO: 396) AD-58682.1 A-119065.1 senseGfsusCfcAfaCfaGfCfAfcCfaAfuAfuCfuUfL96 (SEQ ID NO: 397) A-119067.1antisense asAfsgAfsuAfsuUfgGfugcUfgUfsuGfgAfcsUfsg (SEQ ID NO: 398)AD-58683.1 A-119068.1 sense GsusccAAcAGcAccAAuAucuuL96 (SEQ ID NO: 399)A-119067.1 antisense asAfsgAfsuAfsuUfgGfugcUfgUfsuGfgAfcsUfsg(SEQ ID NO: 400)

Example 3. Efficacy of Si-AAT in Transgenic Mice

Five siRNA duplexes, as described in the preceding examples, with lowIC50 values were tested in vivo for efficacy. The siRNA duplexes wereinjected at 10 mg/kg into transgenic mice expressing the human Z-AATallele, an established model of AAT-deficiency associated liver disease.The mice were dosed on day 0 and serum human AAT was followed for 21days post dose (FIG. 2A). Each point represents an average of three miceand the error bars reflect the standard deviation. The mice weresacrificed on day 21 and their livers were processed to measure mRNAlevels. The graph shows hAAT mRNA normalized to GAPDH for each group(FIG. 2B). The bars reflect the average and the error bars reflect thestandard deviation. As indicated in FIGS. 2A and 2B, AD59054 was themost effective in reducing hAAT mRNA levels in the mice.

Example 4. Durable AAT Suppression in a Dose Responsive Manner

The efficacy of siRNA duplex AD-59054 in the transgenic animal model ofAAT-deficiency associated liver disease was measured by administrationof different doses of siRNA duplex AD-59054 subcutaneously. Serum wasdrawn at different time intervals to measure the serum hAAT proteinlevels using human AAT specific ELISA. The efficacy curve showingmaximum knock-down achieved at different doses tested in mice isdepicted in FIG. 3A. Each point is an average of three animals and theerror bars represent the standard deviation. The duration of knock-downafter a single dose of AAT siRNA at 0.3, 1, 3 or 10 mg/kg is shown inFIG. 3B. Each data point is an average of three animals and the errorbars reflect the standard deviation. The hAAT levels were normalized tothe average of three prebleeds for each animal. The siRNA wasadministered in PBS, hence the PBS group serves as the control toreflect the variability in the serum hAAT levels. Subcutaneousadministration of the AAT siRNA led to dose-dependent inhibition ofserum hATT, with maximum inhibition of >95% observed at a dose of 3mg/kg. A single dose of 1 mg/kg maintained 40% levels of hAAT for atleast 15 days. Animals were also administered AD-59054 at a dose of 0.5mg/kg twice a week (FIG. 3C). The repeat dosing leads to a cumulativeresponse and more than 90% protein suppression. Each data point is anaverage of four animals and the error bars reflect the standarddeviation.

Example 5. Decreased Tumor Incidence with Reduction in Z-AAT

Transgenic human Z-AAT expressing mice develop tumors with age. Thisexperiment was designed to determine whether chronic dosing of theseaged mice with an siRNA of the invention can decrease the tumorincidence in the mice. Specifically, aged mice (25-46 weeks of age) withfibrotic livers were chronically dosed with siRNA duplex AD-58681 todecrease liver tumor incidence. Animals were dosed subcutaneously onceevery other week (Q2W) with PBS or 10 mg/kg AAT siRNA for 11 doses andsacrificed 7 days after the last dose (FIG. 4A). The liver levels ofhAAT mRNA, Col1a2 mRNA and PtPrc mRNA in control and treated groups weremeasured. The AAT siRNA treated animals showed a higher than 90%decrease in hAAT mRNA levels (FIG. 4B). Col1a2 mRNA was measured as amarker of fibrosis and the levels of this marker decreased in AAT siRNAtreated animals (FIG. 4C). PtPrc (CD45) mRNA was measured as a markerfor the presence of immune cells (FIG. 4D). There is more immune cellinfiltration in diseased livers and, as shown in FIG. 4D, the PtPrc mRNAlevels decreased significantly when animals were treated with AAT siRNA.

Serum samples were collected after the first dose to monitor the extentof AAT suppression. All AAT siRNA treated animals showed less than 5%residual AAT protein and a single dose maintained the AAT levels below80% for 14 days before the next dose was administered (FIG. 5A). Table 6provides observations from the animals at the time of sacrifice (day132). Transgenic animals administered the siRNA duplex exhibiteddecreased tumor incidence when compared to untreated control animals.Specifically, four out of six animals treated with PBS showed tumors inthe livers, whereas only one out of six animals treated with AAT siRNAshowed a liver tumor. The p value for the difference in tumor incidencewas calculated by t-test to be 0.045. FIG. 5B and FIG. 5C show PASstaining of liver sections from two littermates treated with either PBSor AAT siRNA. The darker colored dots represent the globules or Z-AATaggregates. These data indicate that siRNA duplex is effective indecreasing Z-AAT levels in transgenic mice and the decreased levels ofZ-AAT show a physiological benefit in the form of healthier livers.

TABLE 6 Treatment Animal # Observation PBS 4734 pale liver 4737 largetumor in left lateral lobe, ~5 mm diameter 4754 pale liver, 2 mm tumorin caudate lobe, many lesions in 2nd aux lobe 4759 dark liver, 1.5 mmtumor in caudate lobe, 1 mm lesion in right medial lobe, multiple 1 mmlesions in 1st aux lobe 4771 3 mm tumor in left lateral lobe 4775 darkliver AAT-siRNA 4748 dark liver 4756 pale liver, 3 mm tumor in caudatelobe 4760 dark liver 4770 nothing abnormal 4772 nothing abnormal 4776nothing abnormal

Example 6. Lead Optimization of AD-59054

As described above, AD-59054 was demonstrated to durably suppress AAT ina dose-responsive manner in vivo. However, the nucleotide sequence ofAD-59054 spans a region in AAT mRNA that includes a prevalent singlenucleotide polymorphism (SNP) (Reference SNP Accession No.: rs1303 (see,e.g., www.ncbi.nlm.nih.gov/projects/SNP)). Specifically, the SNPlocation corresponds to the nucleotide at position 6 (5′ to 3′) in theantisense strand of AD-59054 (i.e., within the seed region of AD-59054).Accordingly, as mismatches within the seed region may lead to off-targeteffects and/or loss of efficacy, additional duplexes having variousbases at position 6 (5′ to 3′) of the antisense strand were preparedbased on the sequence of AD-59054. The target mRNA carries an Acorresponding to position 6 (5′ to 3′) of the antisense strand ofAD-59054. The sequences of these duplexes are provided in Table 7. Table8 provides the sequences of these same duplexes having various chemicalmodifications and conjugated with a trivalent GalNAc.

These modified duplexes were evaluated for efficacy in a single dosefree uptake screen in primary mouse hepatocytes (Hep3B), as describedabove. Hep3B cell mRNA carries a C at the position corresponding toposition 6 (5′ to 3′) of the antisense strand of AD-59054. The IC₅₀values for the duplexes are shown in Table 8. Surprisingly, asdemonstrated therein, a single mismatch within the seed region atposition 6 was tolerated for all bases except C.

A subset of these duplexes was also evaluated for in vivo efficacy.Transgenic mice expressing the human Z-AAT allele (and having an A inthe mRNA corresponding to position 6 (5′ to 3′) of the antisense strandof AD-59054) were injected with 1.0 mg/kg of AD-59054, AD-61719,AD-61700, AD-61726, or AD-61704 on day 0 and serum human AAT, measuredas described above, was followed for 14 days post dose (FIG. 6). Eachpoint represents an average of three mice and the error bars reflect thestandard of deviation. As demonstrated in FIG. 6, AD-61719 and AD-61704perform as well as the parent AD-59054.

TABLE 7 SEQ SEQ Duplex ID ID Name Sense (5′->3) NO: Antisense (5′->3′)NO: AD-59054 CUUCUUAAUGAUUGAACAAAA 401 UUUUGUUCAAUCAUUAAGAAGAC 409AD-61704 CUUCUUAAUGAUUGACCAAAA 402 UUUUGGUCAAUCAUUAAGAAGAC 410 AD-61708CUUCUUAAUGAUUGAUCAAAA 403 UUUUGAUCAAUCAUUAAGAAGAC 411 AD-61712CUUCUUAAUGAUUGAGCAAAA 404 UUUUGCUCAAUCAUUAAGAAGAC 412 AD-61719CUUCUUAAUGAUUGACCAAAA 405 UUUUGIUCAAUCAUUAAGAAGAC 413 AD-61700CUUCUUAAUGAUUGACCAAAA 406 UUUUGNUCAAUCAUUAAGAAGAC 414 AD-61726CUUCUUAAUGAUUGAACAAAA 407 UUUUGNUCAAUCAUUAAGAAGAC 415 AD-61716CUUCUUAAUGAUUGAACAAAA 408 UUUUGNUCAAUCAUUAAGAAGAC 416

Example 7. Lead Optimization of AD-59054

Additional duplexes were prepared based on the sequence of AD-59054,including AD-61444. The modified and unmodified sense and antisensesequences of AD-61444 are provided in Table 9.

TABLE 9 Duplex Unmodified Sense Unmodified Antisense Name (5′ -> 3′) (5′-> 3′) AD-61444 CUUCUUAAUGAUUGAACAAAA UUUUGUUCAAUCAUUAAGAAGAC(SEQ ID NO: 417) (SEQ ID NO: 419) Modified Sense (5′ -> 3′)Modified Antisense (5′ -> 3′) csusucuuaauGfAfuugaacaaaaL96usUfsuUfgUfuCfaAfucaUfuAfaGfaAfgsasc (SEQ ID NO: 418) (SEQ ID NO: 420)

TABLE 8 SEQ SEQ Duplex Base at  ID ID IC₅₀ Name position 6 Sense (5′ ->3′) NO: Antisense (5′ -> 3′) NO: mean AD-59054 U (parentCfsusUfcUfuAfaUfGfAfuUfgAfaCfaAfaAfL96 421usUfsuUfgUfuCfaAfucaUfuAfaGfaAfgsasc 429 0.098 compound) AD-61704 GCfsusUfcUfuAfaUfGfAfuUfgAfcCfaAfaAfL96 422usUfsuUfgGfuCfaAfucaUfuAfaGfaAfgsasc 430 0.102 AD-61708 ACfsusUfcUfuAfaUfGfAfuUfgAfuCfaAfaAfL96 423usUfsuUfgAfuCfaAfucaUfuAfaGfaAfgsasc 431 0.147 AD-61712 CCfsusUfcUfuAfaUfGfAfuUfgAfgCfaAfaAfL96 424usUfsuUfgCfuCfaAfucaUfuAfaGfaAfgsasc 432 1.499 AD-61719 I (inosine)CfsusUfcUfuAfaUfGfAfuUfgAfcCfaAfaAfL96 425usUfsuUfgiuCfaAfucaUfuAfaGfaAfgsasc 433 0.088 AD-61700 dICfsusUfcUfuAfaUfGfAfuUfgAfcCfaAfaAfL96 426usUfsuUfgdIuCfaAfucaUfuAfaGfaAfgsasc 434 0.097 (deoxyinosine)(S/AS¹: C/dI) AD-61726 dI CfsusUfcUfuAfaUfGfAfuUfgAfaCfaAfaAfL96 427usUfsuUfgdIuCfaAfucaUfuAfaGfaAfgsasc 435 0.059 (deoxyinosine)(S/AS: A/dI) AD-61716 abasic 2′-OMeCfsusUfcUfuAfaUfGfAfuUfgAfaCfaAfaAfL96 428usUfsuUfgY34uCfaAfucaUfuAfaGfaAfgsasc 436 0.333 ¹ S/AS: Sense/Antisense.

Example 8. Non-Human Primate Dosing of AD-59054, AD-61719, and AD-61444

AD-59054, AD-61719, and AD-61444 were tested for efficacy in non-humanprimates by administering to the primates a single dose of 1 mg/kg or 3mg/kg of AD-59054, AD-61719, or AD-61444. Serum samples were collectedfive days prior to administration, at day 0, and at days 3, 7, 10, 15,20, and 30 after administration to monitor the extent of AAT suppressionby measuring serum hAAT protein levels using human AAT specific ELISA.There were no changes in cytokine or chemokine levels in the serum ofthe animals administered any of the compounds, and no injection sitereactions or drug related health concerns were associated withadministration of the compounds. FIG. 7 shows that a single dose of 1mg/kg of AD-59054, AD-61719, or AD-61444 (7A) or a single dose of 3mg/kg of AD-59054, AD-61719, or AD-61444 (7B) results in a dosedependent and durable lowering of AAT protein.

We claim:
 1. A double stranded ribonucleic acid (dsRNA) agent forinhibiting expression of Serpina1 in a cell, wherein said dsRNA agentcomprises a sense strand and an antisense strand forming adouble-stranded region, wherein said sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, and said antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from any one of the nucleotide sequences of SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25,wherein substantially all of the nucleotides of said sense strand andsubstantially all of the nucleotides of said antisense strand aremodified nucleotides, and wherein said sense strand is conjugated to aligand attached at the 3′-terminus.
 2. The dsRNA agent of claim 1,wherein one of the 3 nucleotide differences in the nucleotide sequenceof the antisense strand is a nucleotide mismatch in the seed region ofthe antisense strand.
 3. The dsRNA agent of claim 1, wherein said sensestrand and said antisense strand comprise a region of complementaritywhich comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from any one of the sequences listed in any one ofTables 1, 2, 5, 7, 8, and
 9. 4. A double stranded ribonucleic acid(dsRNA) agent capable of inhibiting the expression of Serpina1 in acell, wherein said dsRNA agent comprises a sense strand substantiallycomplementary to an antisense strand, wherein said antisense strandcomprises a region substantially complementary to part of an mRNAencoding Serpina1, wherein each strand is about 14 to about 30nucleotides in length, wherein said dsRNA agent is represented byformula (III): (III) sense: 5′n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′

wherein: i, j, k, and l are each independently 0 or 1; p, p′, q, and q′are each independently 0-6; each N_(a) and N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 0-25 nucleotides whichare either modified or unmodified or combinations thereof, each sequencecomprising at least two differently modified nucleotides; each N_(b) andN_(b)′ independently represents an oligonucleotide sequence comprising0-10 nucleotides which are either modified or unmodified or combinationsthereof; each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or maynot be present, independently represents an overhang nucleotide; XXX,YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent onemotif of three identical modifications on three consecutive nucleotides;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and whereinthe sense strand is conjugated to at least one ligand.
 5. The dsRNAagent of claim 4, wherein Na′ comprises 1-25 nucleotides, and whereinone of the 1-25 nucleotides at one of positions 2-9 from the 5′ end is anucleotide mismatch.
 6. The dsRNA agent of claim 1 or 4, wherein theligand is one or more GalNAc derivatives attached through a bivalent ortrivalent branched linker.
 7. The dsRNA agent of claim 1 or 4, whereinsaid agent further comprises at least one phosphorothioate ormethylphosphonate internucleotide linkage.
 8. The dsRNA agent of claim4, wherein the Y nucleotides contain a 2′-fluoro modification.
 9. ThedsRNA agent of claim 4, wherein the Y′ nucleotides contain a 2′-O-methylmodification.
 10. The dsRNA agent of claim 1 or 4, wherein said RNAiagent is selected from the group of RNAi agents listed in any one ofTables 1, 2, 5, 7, 8, and
 9. 11. The dsRNA agent of claim 1 or 4,wherein said RNAi agent is selected from the group consisting ofAD-58681, AD-59054, AD-61719, and AD-61444.
 12. A cell containing thedsRNA agent of claim 1 or
 9. 13. A composition comprising a modifiedantisense polynucleotide agent, wherein said agent is capable ofinhibiting the expression of Serpina1 in a cell, and comprises asequence complementary to a sense sequence selected from the group ofthe sequences listed in any one of Tables 1, 2, 5, 7, 8, and 9, whereinthe polynucleotide is about 14 to about 30 nucleotides in length.
 14. Apharmaceutical composition comprising the dsRNA agent of claim 1 or 4.15. A method of inhibiting Serpina1 expression in a cell, the methodcomprising: (a) contacting the cell with the dsRNA agent of claim 1 or4, or the pharmaceutical composition of claim 14; and (b) maintainingthe cell produced in step (a) for a time sufficient to obtaindegradation of the mRNA transcript of a Serpina1 gene, therebyinhibiting expression of the Serpina1 gene in the cell.
 16. A method oftreating a subject having a Serpina1 deficiency variant-associatedassociated disease, comprising administering to the subject atherapeutically effective amount of the dsRNA agent of claim 1 or 4, orthe pharmaceutical composition of claim 14, thereby treating saidsubject.
 17. A method of treating a subject having a Serpina1 deficiencyvariant-associated associated disease, comprising subcutaneouslyadministering to the subject a therapeutically effective amount of adouble stranded ribonucleic acid (dsRNA) agent, wherein said dsRNA agentcomprises a sense strand and an antisense strand forming a doublestranded region, wherein said sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, and said antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from any one of the nucleotide sequences of SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25,wherein substantially all of the nucleotides of said antisense strandcomprise a modification selected from the group consisting of a2′-O-methyl modification and a 2′-fluoromodification, wherein saidantisense strand comprises two phosphorothioate internucleotide linkagesat the 5′-terminus and two phosphorothioate internucleotide linkages atthe 3′-terminus, wherein substantially all of the nucleotides of saidsense strand comprise a modification selected from the group consistingof a 2′-O-methyl modification and a 2′-fluoromodification, wherein saidsense strand comprises two phosphorothioate internucleotide linkages atthe 5′-terminus and, wherein said sense strand is conjugated to one ormore GalNAc derivatives attached through a branched bivalent ortrivalent linker at the 3′-terminus, thereby treating the subject. 18.The method of claim 17, wherein the Serpina1 associated disease is aliver disorder.
 19. A method of treating a subject having a Serpina1deficiency variant-associated disorder to reduce the progression of theliver disorder to hepaocellular carcinoma, comprising administering tothe subject a therapeutically effective amount of the dsRNA agent ofclaim 1 or 4, or the pharmaceutical composition of claim 14, therebytreating the subject.
 20. A method of reducing the accumulation ofmisfolded Serpina1 in the liver of a subject having a Serpina1deficiency variant-associated disorder, comprising administering to thesubject a therapeutically effective amount of the dsRNA agent of acclaim1 or 4, or the pharmaceutical composition of claim 14, thereby reducingthe accumulation of misfolded Serpina1 in the liver of the subject.